CN106632678A

CN106632678A - Human antibodies against tissue factor

Info

Publication number: CN106632678A
Application number: CN201610367892.8A
Authority: CN
Inventors: 桑德拉.弗普洛根; 戴维.P.E.萨廷; 勒内.M.A.霍伊特; 保罗.帕伦; 詹.范德温克尔; 维比克.M.布雷恩霍尔特; 伊娃.埃恩鲁思; 奥利.巴德斯加德; 汤姆.文克; 威廉.K.布利克; 米沙.霍特坎普; 马罗斯卡.奥德肖恩; 罗布.N.德琼格
Original assignee: Genmab AS
Current assignee: Genmab AS
Priority date: 2008-12-09
Filing date: 2009-12-09
Publication date: 2017-05-10
Anticipated expiration: 2029-12-09
Also published as: KR101736447B1; EP3159358A1; US9714297B2; JP5860285B2; MX355181B; DK2376537T3; EP3159358B1; JP7069095B2; US20210171657A1; US20160053020A1; IL212856A; US20230416403A1; BRPI0922350B8; JP2022078018A; CA2746101C; JP2020022461A; JP2018038418A; KR101998492B1; JP2012511314A; MX2011005669A

Abstract

Isolated human monoclonal antibodies which bind to human TF and related antibody-based compositions and molecules, are disclosed. Also disclosed are pharmaceutical compositions comprising the antibodies, and therapeutic and diagnostic methods for using the antibodies.

Description

For the human antibody of tissue factor

The application be based on the applying date be on December 9th, 2009, priority date be on December 9th, 2008, Application No. 200980156348.7, it is entitled：The divisional application of the patent application of " for the human antibody of tissue factor ".

Invention field

The present invention relates to tissue factor is directed to, the purposes of the particularly antibody of human tissue factor, and this antibody-like, particularly Their purposes in treating cancer, inflammation and vascular diseases.

Background of invention

Tissue factor (TF), also referred to as factor I (thromboplastin), factor III or CD142, are one The protein being present in subendothelial tissue, blood platelet and leucocyte is planted, is caused from proenzyme factor (prothrombin) Necessary to forming fibrin ferment.Fibrin ferment is formed and ultimately results in blood clotting.Tissue factor enables cell to trigger coagulation cascade, And play the function of proconvertin high-affinity receptor.Resulting complex provides a kind of catalyzed event, and the event is born Duty causes the cascade of blood coagulating protein enzyme by special limited proteolysis (limited proteolysis).With these albumen Other are different as the confactor that nonfunctional precursor forms are circulated in enzyme cascade, and this factor is a kind of initiation of strength Agent (initiator), has completely function when its expression is on cell surface.

Tissue factor is the cell surface receptor of serine protease factor VIIa (FVIIa).FVIIa and tissue factor With reference to have been found to can commencing signal transmittance process in the cell, the signal transfer function plays work in blood vessel generation With.Although blood vessel generation is a kind of normal process in growth and development and wound healing, it is also tumour from not Dormancy state is transformed into a basic step during malignant state：The egg for participating in blood vessel generation can be produced when cancer cell is obtained White matter, i.e., so-called angiogenesis growth factors, ability when, these protein are discharged in tissue around by tumour, and are pierced Swash existing healthy blood vessel to germinate (sprout) new blood vessel towards tumour and enter inside tumor.Once new blood vessel enters tumour Interior, it just can quickly increase its size, and attack the tissue and organ of local.By these new blood vessels, cancer cell can enter One step is escaped in circulation, and inhabits the new tumour (transfer) of formation in other organs.

Additionally, TF plays a role in inflammation.The effect of TF is considered as the (A.J.Chu mediated by blood clotting: “Tissue factor mediates inflammation”in Archives of biochemistry and biophysics,2005,vol.440,No.2,pp.123-132).Therefore, for example, by the anti-TF antibody suppressions TF of monoclonal, It is significant in interference solidification-inflammation circulation, not only facilitate anti-inflammatory and contribute to anti-vascular disease.

TF expression is observed in perhaps eurypalynous cancer, and the disease higher with invasion is relevant.And, people TF is also with a kind of presence of alternative splice forms, i.e. asHTF.Recently, it has been found that asHTF promotes tumour growth (Hobbs et al.2007Thrombosis Res.120(2)S13-S21)。

The antibody combined with TF is had been disclosed in the prior art：

WO98/40408 is disclosed individualism or can be present in TF with reference to the antibody of natural (native) people TF: In VIIa complexs, can effectively prevent factor X from being attached to TF or the complex, blood coagulation is reduced whereby.The document discloses this to resist Body can be used for mitigate invasive medical procedure (invasive medical procedure), such as artery or openheart surgery, after Thrombosis, or eliminate due to the blood coagulation caused using medical measure (medical implementation).Other resist Body is disclosed for in-vivo diagnostic method, including the diagnostic imaging in vivo of natural human TF.

WO04/094475 provides the antibody that can be combined with human tissue factor, and it does not press down compared with normal plasma control The blood coagulation of factor mediation processed.Human antibody is not described.It is said that the antibody can be used for treating cancer.

WO03/093422 is related to and TF:The binding affinity of VIIa complexs is more than and the binding affinity of TF itself Antibody.And propose purposes of these antibody as anti-coagulants in some disease treatments, such as septicemia, dispersivity Ink vessel transfusing Coagulopathy in blood coagulation, cerebral arterial thrombosis, thrombosis, acute coronary syndrome and TCA.

WO01/27079 discloses composition and method, for suppressing abnormal cell proliferation, particularly endothelial cell proliferation, Such as cancer, embryo's anormogenesis, immune response malfunction, and the blood vessel relevant with neovascularization and tumour growth are sent out It is raw.There is provided many active materials, including antibody, but specific antibody is not disclosed.

WO03/037361 is related to TF activators or antagonist is used for the purposes of the related treatment of Apoptosis.

WO03/029295 is related to detached human antibody, itself and people's TF immune responses, suppresses the combination of proconvertin a. However, this application does not disclose any example of the antibody with these properties.

There are some monoclonal antibody therapies to be approved for treating different types of tumour, including such as bevacizumab (bevacizumab)Cetuximab (cetuximab)Victibix (panitumumab) (Vectibix^TM) and trastuzumab (trastuzumab)

Brief summary of the invention

Although having been achieved for many progress, remaining a need for the improved method based on therapeutic antibodies is used to treat Serious disease, such as improved treatment of cancer.

It is an object of the present invention to provide for the new high specific of medical application, the anti-TF antibody of effective people.This Invention antibody shows the TF binding characteristics different from the antibody having been described in this area.In preferred embodiments, the present invention Antibody on human tissue factor there is high-affinity, the cytotoxicity (ADCC) that mediate antibody is relied on suppresses FVIIa to combine TF, Suppress ERK phosphorylations and the IL-8 releases of FVIIa inductions, do not suppress or weaker suppress blood coagulation.

The present invention includes following item：

1. a kind of human antibody of combination human tissue factor.

2. 1 antibody, wherein when being measured with the assay method described in embodiment 13, the antibody with 3nM or Lower, such as 0.50nM or lower, such as 0.35nM or lower, such as 0.20nM or lower, such as 0.1nM or lower are apparent Affinity (EC₅₀) combined with the extracellular domain of tissue factor.

3. the antibody of any one of aforementioned item, the wherein antibody combine the mammalian cell of the expression tissue factor, for example The A431 cells transfected with the construct of encoding tissue factor, it is preferable that when being carried out with the assay method described in embodiment 14 During measure, its apparent affinity (EC₅₀) it is 10nM or lower, such as 8nM or lower, such as 5nM or lower, such as 2nM or more It is low, such as 1nM or lower, such as 0.5nM or lower, such as 0.3nM or lower.

4. the cell that the antibody of any one of aforementioned item, the wherein antibody can induce antibody-dependant in A431 cells is thin Cellular toxicity, it is preferable that when being measured with the assay method described in embodiment 20, its EC₅₀It is worth for 2nM or lower, for example 1nM or lower, such as 0.7nM or lower, or 0.3nM or lower, such as 0.20nM or lower, or 0.1nM or lower, or Person 0.05nM or lower.

5. the antibody of any one of aforementioned item, wherein when being measured with the method described in embodiment 24, the antibody Can effectively suppress the growth of MDA-MB-231 tumours set up, and/or when being measured with the method described in embodiment 26 When, can effectively suppress the growth of BxPC3 tumours set up.

6. the antibody of any one of aforementioned item, the wherein blood coagulation of antibody suppression tissue factor induction, it is preferable that when with reality When applying the assay method described in example 19 and being measured, its median inhibitory concentration is less than 10nM, e.g., less than 5nM, e.g., less than 2nM, e.g., less than 1nM.

7. the antibody of any one of aforementioned item, wherein antibody suppression FVIIa is combined with tissue factor, it is preferable that when with When assay method described in embodiment 15 is measured, the maximum suppression value that it suppresses is more than 80%, is greater than 90%.

8. the antibody of any one of aforementioned item, the IL-8 of the MDA-MB-231 cells of wherein antibody suppression FVIIa inductions Release, it is preferable that when being measured with the assay method described in embodiment 17, the maximum suppression value of suppression is more than 40%, 50% is greater than, 60% is greater than.

9. the antibody in any one of aforementioned item, wherein antibody suppression FX is transformed into FXa by TF/FVIIa complexs, excellent Selection of land, when being measured with the assay method described in embodiment 18, suppresses to be less than 50%, e.g., less than 40%, for example, exist In the range of 1-30%.

10. the antibody of any one of aforementioned item, wherein the antibody with include comprising sequence SEQ ID NO:9 VH areas and Comprising sequence SEQ ID NO:The antibody competition tissue factor in 65 VL areas is combined.

The antibody of any one of 11. aforementioned items, wherein and the combination of tissue factor be not related to the following arbitrary of tissue factor Amino acid：The W of 45, the Y of K or 94 of 46.

The antibody of any one of 12. aforementioned items, wherein the antibody with include comprising sequence SEQ ID NO:37 VH areas And comprising sequence SEQ ID NO:The antibody competition tissue factor in 93 VL areas is combined.

The antibody of any one of 13. aforementioned items, wherein the ERK phosphorylations of antibody suppression FVIIa inductions, it is preferable that When being measured with the assay method described in embodiment 16, its median inhibitory concentration is less than 10nM, e.g., less than 5nM, example Such as less than 2nM.

The antibody of 14. any one of 1-7 and 9-13, wherein antibody suppression ERK phosphorylations, it is preferable that when with enforcement When assay method described in example 16 is measured, its median inhibitory concentration is less than 10nM, e.g., less than 5nM, e.g., less than 2nM, and the suppression of the IL-8 releases to FVII inductions as described in the assay method in embodiment 17 is less than maximum 10%.

The antibody of any one of 15. aforementioned items, wherein antibody can induce C3c and C4c depositions, it is preferable that such as embodiment It is measured described in 21, the antibody can induce C3c and C4c depositions.

The antibody of any one of 16. aforementioned items, wherein as described in Example 28, Fab fragments are extracellular with tissue factor Domain combines, and the EC50 values measured with ELISA are less than 0.1 μ g/mL, such as less than such as less than 0.05 μ g/mL, 0.04 μ g/mL.

The antibody of any one of 17. 1-15, wherein as described in Example 28, Fab fragments are extracellular with tissue factor Domain combines, and the EC50 values measured according to ELISA are higher than 1.0 μ g/mL.

The antibody of any one of 18. 1-15, wherein as described in Example 28, Fab fragments are extracellular with tissue factor Domain combines, and EC50 values are less than 10 μ g/mL, such as less than such as less than 1 μ g/mL, 0.5 μ g/mL, or less than 0.2 μ g/mL.

The antibody of 19. any of the above-described, the wherein antibody are combined with human tissue factor but not combined with the rat tissue factor, and And compared to and people TF combination, itself and reorganization construct 42-84mm show the combination for reducing, and the reorganization construct is comprising people TF sequences, simply amino acid 42-84 replaced by mouse sequence, as described in Example 27.

The antibody of 20. 1-18, the wherein antibody are combined with human tissue factor but not combined with the rat tissue factor, and phase Than in and people TF combination, itself and reorganization construct 85-122mm show reduce combination, the reorganization construct comprising people TF Sequence, simply amino acid 85-122 replaced by mouse sequence, as described in Example 27.

The antibody of 21. 1-18, the wherein antibody are combined with human tissue factor but not combined with the rat tissue factor, and phase Than in and people TF combination, itself and reorganization construct 123-137mm show reduce combination, the reorganization construct comprising people TF Sequence, simply amino acid/11 23-137 replaced by mouse sequence, as described in Example 27.

The antibody of 22. 1-18, the wherein antibody are combined with human tissue factor but not combined with the rat tissue factor, and phase Than in and people TF combination, itself and reorganization construct 185-225mm show reduce combination, the reorganization construct comprising people TF Sequence, simply amino acid/11 85-225 replaced by mouse sequence, as described in Example 27.

The antibody of 23. 1-18, the wherein antibody are combined with human tissue factor but not combined with the rat tissue factor, and phase Than in and people TF combination, itself and reorganization construct 226-250mm show reduce combination, the reorganization construct comprising people TF Sequence, simply amino acid 226-250 replaced by mouse sequence, as described in Example 27.

24. according to the antibody of any one of item 19-23, wherein compared to and people TF combination, itself and the structure of the reorganization more than a kind Build body show reduce combination, for example and it is following reorganization construct reduction combination：

Such as reorganization construct 42-84mm defined in item 19 and such as reorganization construct 85- defined in item 20 122mm, or

Such as reorganization construct 123-137mm defined in item 21, and such as reorganization construct 185- defined in item 22 225mm, and such as reorganization construct 226-250mm defined in item 23.

Antibody in any one of 25. aforementioned items, wherein the antibody

- and include comprising sequence SEQ ID NO:9 VH areas and comprising sequence SEQ ID NO:The antibody in 65 VL areas is competing Tissue factor combination is striven, and

- not with include comprising sequence SEQ ID NO:37 VH areas and comprising sequence SEQ ID NO:The antibody in 93 VL areas Competition tissue factor is combined.

The antibody of 26. 25, wherein the antibody includes such VH CDR3 areas, it has

A) sequence as follows

-SEQ ID No:12,

-SEQ ID No:16,

-SEQ ID No:20,

-SEQ ID No:24,

-SEQ ID No:28,

Or

B) variant of any sequence, such as it is amino acid modified with most 1,2,3,4 or 5, it is preferred to replace for example The conservative variant replaced.

The antibody of 27. 26, the wherein antibody include thering is such as SEQ ID NO:The VH of sequence or its variant shown in 12 CDR3 areas, the wherein variant one or more positions in position 2,3,6,9 and 11 include modification, it is preferable that wherein this is repaiied Decorations are to replace, it is highly preferred that wherein the replacement is selected from the group：

A) R at position 2 is replaced by K,

B) S at position 3 is replaced by A or T,

C) G at position 6 is replaced by T,

D) L at position 9 is replaced by F, and

E) S at position 11 is replaced by Y.

28. 1 or 26 antibody, the wherein antibody includes：

A) CDR1,2 and 3 sequence SEQ ID NO is included:10,11 and 12 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:66,67 and 68 VL areas,

B) CDR1,2 and 3 sequence SEQ ID NO is included:14,15 and 16 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:70,71 and 72 VL areas,

C) CDR1,2 and 3 sequence SEQ ID NO is included:18,19 and 20 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:74,75 and 76 VL areas,

D) CDR1,2 and 3 sequence SEQ ID NO is included:22,23 and 24 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:78,79 and 80 VL areas,

E) CDR1,2 and 3 sequence SEQ ID NO is included:26,27 and 28 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:82,83 and 84 VL areas, or

F) variant of arbitrary antibody, wherein the variant has most 1,2 or 3 ammonia preferably in the sequence Base acid is modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 29. 26, comprising such VH, its

A) and selected from SEQ ID NO:9,13,17,21 and 25 VH region sequences have at least 80% homogeneity, for example, at least 90%, at least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:9,13,17,21, the 21 VH region sequences with 25 are compared with most 20, such as 15 It is individual, or 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 30. 26, comprising such VL, its

A) and selected from SEQ ID NO:65,69,73,77 and 81 VL region sequences have at least 80% homogeneity, for example extremely Few 90%, at least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:65,69,73, the 77 VH region sequences with 81 are compared with most 20, such as 15, Or 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 31. 26, including：

A) sequence SEQ ID NO are included:9 VH areas and comprising sequence SEQ ID NO:65 VL areas,

B) sequence SEQ ID NO are included:13 VH areas and comprising sequence SEQ ID NO:69 VL areas,

C) sequence SEQ ID NO are included:17 VH areas and comprising sequence SEQ ID NO:73 VL areas,

D) sequence SEQ ID NO are included:21 VH areas and comprising sequence SEQ ID NO:77 VL areas,

E) sequence SEQ ID NO are included:25 VH areas and comprising sequence SEQ ID NO:81 VL areas, or

F) variant of arbitrary antibody, wherein the variant preferably has most 1,2 or 3 ammonia in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of any one of 32. 1-24, wherein the antibody

- and include comprising sequence SEQ ID NO:37 VH areas and comprising sequence SEQ ID NO:The antibody in 93 VL areas is competing Strive tissue factor combination.

The antibody of 33. 32, wherein the antibody includes such VH CDR3 areas, it has

A) sequence as follows

-SEQ ID No:8,

-SEQ ID No:52,

Or

B) variant of any sequence, such as amino acid modified with most 1,2 or 3, preferred replacement is for example guarded The variant of replacement.

34. 1 or 32 antibody, the wherein antibody includes：

A) CDR1,2 and 3 sequence SEQ ID NO is included:6,7 and 8 VH areas, and comprising CDR1,2 and 3 sequence SEQ ID NO:62,63 and 64 VL areas,

B) CDR1,2 and 3 sequence SEQ ID NO is included:50,51 and 52 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:106,107 and 108 VL areas, or

C) variant of arbitrary antibody, wherein the variant has most 1,2 or 3 ammonia preferably in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 35. 32, including such VH, its

A) and selected from SEQ ID NO:5 and 49 VH region sequences have at least 80% homogeneity, for example, at least 90%, at least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:The 5 VH region sequences with 49 are compared with most 20, such as 15, or 10, Or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 36. 32, it includes such VL, the VL

A) and selected from SEQ ID NO:61 and 105 VL region sequences have at least 80% homogeneity, for example, at least 90%, extremely Few 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:The 61 VH region sequences with 105 are compared with most 20, such as 15, or 10 It is individual, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 37. 32, it includes：

A) sequence SEQ ID NO are included:5 VH areas and comprising sequence SEQ ID NO:61 VL areas,

B) sequence SEQ ID NO are included:49 VH areas and comprising sequence SEQ ID NO:105 VL areas, or

C) variant of arbitrary antibody, wherein the variant preferably has most 1,2 or 3 ammonia in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of any one of 38. 1-24, wherein the antibody

- not with include comprising sequence SEQ ID NO:9 VH areas and comprising sequence SEQ ID NO:The antibody in 65 VL areas Competition tissue factor is combined, and

The antibody of 39. 38, wherein the antibody includes such VH CDR3 areas, it has

A) sequence as follows

-SEQ ID No:32,

-SEQ ID No:36,

-SEQ ID No:40,

-SEQ ID No:56,

Or

B) variant of any sequence, such as it is amino acid modified with most 1,2 or 3, it is preferred to replace, for example guard The variant of replacement.

40. 1 or 38 antibody, the wherein antibody includes：

A) CDR1,2 and 3 sequence SEQ ID NO is included:30,31 and 32 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:86,87 and 88 VL areas,

B) CDR1,2 and 3 sequence SEQ ID NO is included:34,35 and 36 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:90,91 and 92 VL areas,

C) CDR1,2 and 3 sequence SEQ ID NO is included:38,39 and 40 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:94,95 and 96 VL areas,

D) CDR1,2 and 3 sequence SEQ ID NO is included:54,55 and 56 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:110,11 and 112 VL areas, or

E) variant of arbitrary antibody, wherein the variant has most 1,2 or 3 ammonia preferably in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 41. 38, it includes such VH, the VH

A) and selected from SEQ ID NO:29,33,37 and 53 VH region sequences have at least 80% homogeneity, for example, at least 90%, at least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:29,33, the 37 VH region sequences with 53 are compared with most 20, such as 15, or Person 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 42. 38, it includes such VL, the VL

A) and selected from SEQ ID NO:85,89,93 and 109 VH region sequences have at least 80% homogeneity, for example, at least 90%, at least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:85,89, the 93 VH region sequences with 109 are compared with most 20, such as 15, or Person 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 43. 38, it includes：

A) sequence SEQ ID NO are included:29 VH areas and comprising sequence SEQ ID NO:85 VL areas,

B) sequence SEQ ID NO are included:33 VH areas and comprising sequence SEQ ID NO:89 VL areas,

C) sequence SEQ ID NO are included:37 VH areas and comprising sequence SEQ ID NO:93 VL areas,

D) sequence SEQ ID NO are included:53 VH areas and comprising sequence SEQ ID NO:109 VL areas, or

E) variant of arbitrary antibody, wherein the variant preferably has most 1,2 or 3 ammonia in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of any one of 44. aforementioned 1-24, wherein the antibody with include comprising sequence SEQ ID NO:41 VH areas and comprising sequence SEQ ID NO:The antibody competition tissue factor in 97 VL areas is combined.

The antibody of 45. 44, wherein the antibody includes such VH CDR3 areas, it has

A) sequence as follows

-SEQ ID No:4,

-SEQ ID No:44,

-SEQ ID No:48,

Or

46. 1 or 44 antibody, the wherein antibody includes：

A) CDR1,2 and 3 sequence SEQ ID NO is included:2,3 and 4 VH areas, and comprising CDR1,2 and 3 sequence SEQ ID NO:58,59 and 60 VL areas,

B) CDR1,2 and 3 sequence SEQ ID NO is included:42,43 and 44 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:98,99 and 100 VL areas,

C) CDR1,2 and 3 sequence SEQ ID NO is included:46,47 and 48 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:102,103 and 104 VL areas, or

D) variant of arbitrary antibody, wherein the variant has most 1,2 or 3 ammonia preferably in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 47. 44, it includes such VH, the VH

A) and selected from SEQ ID NO:The VH region sequences of Isosorbide-5-Nitrae 1 and 45 have at least 80% homogeneity, for example, at least 90%, At least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:VH region sequence of the Isosorbide-5-Nitrae 1 with 45 is compared with most 20, such as 15, or 10 It is individual, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 48. 44, it includes such VL, the VL

A) and selected from SEQ ID NO:57,97 and 101 VL region sequences have at least 80% homogeneity, for example, at least 90%, at least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:57, the 97 VH region sequences with 101 are compared with most 20, such as 15, or 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of 49. 44, including：

A) sequence SEQ ID NO are included:1 VH areas and comprising sequence SEQ ID NO:57 VL areas,

B) sequence SEQ ID NO are included:41 VH areas and comprising sequence SEQ ID NO:97 VL areas,

C) sequence SEQ ID NO are included:45 VH areas and comprising sequence SEQ ID NO:101 VL areas, or

D) variant of arbitrary antibody, wherein the variant preferably has most 1,2 or 3 ammonia in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

The antibody of any one of 50. 1-5, wherein when being measured with the method described in embodiment hereof 22, should Antibody is less than 5nM, e.g., less than e.g., less than 3.5nM, 2nM to the affinity of tissue factor.

The antibody of any one of 51. 1-5, wherein when being measured with the method described in embodiment hereof 22, should The kd of antibody is more than 10^-3sec^-1, and/or when being measured with the method described in embodiment hereof 22, the ka of the antibody is big In 5x10⁴mol^-1sec^-1。

The antibody of any one of 52. 1-5, wherein when being measured with the affinity method described in embodiment hereof 22 When, the kd of the antibody is more than 10^-3sec^-1, and when being measured with the affinity method described in embodiment hereof 22, should The affinity of antibody is less than 5nM, e.g., less than e.g., less than 1nM, 0.2nM.

53. 1-5 or 51 or any one of 52 antibody, the wherein antibody do not show combination with health tissues, particularly Combination is not shown with people's glomerulus, for example, is measured with the assay method described in embodiment 23；But show with pancreatic neoplasm and tie Close, for example, measured with the assay method described in embodiment hereof 23.

The antibody of 54. any one of 1-5 or 51-53, wherein when being surveyed with the method described in embodiment hereof 26 Regularly, the antibody can effectively suppress the growth of the BX-PC3 tumours set up.

The antibody of any one of 55. aforementioned items, the wherein antibody further have one or more following property：Suppress to increase Grow, suppress tumor vessel generation, inducing apoptosis of tumour cell to be combined with the tissue factor of alternative splicing.

The antibody of any one of 56. 1-5, its with combined comprising following antibody competition tissue factor：

B) sequence SEQ ID NO are included:1 VH areas and comprising sequence SEQ ID NO:57 VL areas,

C) sequence SEQ ID NO are included:5 VH areas and comprising sequence SEQ ID NO:61 VL areas,

D) sequence SEQ ID NO are included:13 VH areas and comprising sequence SEQ ID NO:69 VL areas,

E) sequence SEQ ID NO are included:17 VH areas and comprising sequence SEQ ID NO:73 VL areas,

F) sequence SEQ ID NO are included:21 VH areas and comprising sequence SEQ ID NO:77 VL areas,

G) sequence SEQ ID NO are included:25 VH areas and comprising sequence SEQ ID NO:81 VL areas,

H) sequence SEQ ID NO are included:29 VH areas and comprising sequence SEQ ID NO:85 VL areas,

I) sequence SEQ ID NO are included:33 VH areas and comprising sequence SEQ ID NO:89 VL areas,

J) sequence SEQ ID NO are included:37 VH areas and comprising sequence SEQ ID NO:93 VL areas,

K) sequence SEQ ID NO are included:41 VH areas and comprising sequence SEQ ID NO:97 VL areas,

L) sequence SEQ ID NO are included:45 VH areas and comprising sequence SEQ ID NO:101 VL areas,

M) sequence SEQ ID NO are included:49 VH areas and comprising sequence SEQ ID NO:105 VL areas, or

N) sequence SEQ ID NO are included:53 VH areas and comprising sequence SEQ ID NO:109 VL areas.

The antibody of any one of 57. 1-5, its with the same epitope on following antibody bind tissue factors：

The antibody of any one of 58. aforementioned items, the wherein antibody include：

- it is derived from people's germline V being selected from the group_HThe weight chain variable district of sequence：IGHV1-18*01,IGHV3-23*01, IGHV3-30*01, IGHV3-33*01, IGHV3-33*03, IGHV1-69*02, IGHV1-69*04 and IGHV5-51*01, and/ Or

- it is derived from people's germline V being selected from the group_κThe light chain variable district of sequence：IGKV3-20*01,IGKV1-13*02, IGKV3-11*01 and IGKV1D-16*01.

The antibody of any one of 59. aforementioned items, the wherein antibody are full length antibodies, preferred IgG1 antibody, particularly IgG1, κ antibody.

The antibody of any one of 60. aforementioned items, the wherein antibody coupling are in another part, such as cytotoxic moiety, radiation Property isotope or medicine.

The antibody of 61. any one of 1-3 or 15-49, the wherein antibody are effector function defect antibody.

The antibody of 62. 61, wherein the effector function defect anti-tissue factor antibodies are the antibody of stabilized human IgG 4, For example wherein 4 CH4 of human IgG, 09 arginine is replaced by lysine, threonine, methionine or leucine, excellent Lysine is selected, and/or wherein hinge area includes the antibody of Cys-Pro-Pro-Cys sequences.

The antibody of 63. any one of 1-3 or 15-49, the wherein antibody are univalent antibodies.

The antibody of 64. 63, wherein the univalent antibody builds with the following method, it includes：

I) nucleic acid construct of the coding univalent antibody light chain is provided, the construct is special comprising the selected antigen of coding The nucleotide sequence in the VL areas of heterogenetic antibody and the nucleotide sequence in the constant CL areas of coding Ig, wherein the coding is selected resisting The nucleotide sequence in the CL areas of the nucleotide sequence in the VL areas of former specific antibody and the coding Ig is operatively coupled on one Rise, and wherein, in the case of IgG1 hypotypes, encode the nucleotide sequence in the CL areas through modification so that the CL areas Any amino acid as described below is not contained：Under conditions of it there is polyclonal human IgG or when animals or humans is applied to, The amino acid can form disulfide bond or covalent bond with other comprising with the peptide of the CL areas identical amino acid sequence；

Ii the nucleic acid construct of the coding univalent antibody heavy chain) is provided, the construct includes the selected antigen of coding The nucleotide sequence in the VH areas of specific antibody and the nucleotide sequence in the constant CH areas of encoding human Ig, wherein the core in coding CH areas Nucleotide sequence is through modification so that corresponding to the region of hinge area, and other regions in the CH areas as required by Ig hypotypes, such as CH3 areas, do not contain any amino acid residue as described below：It is under conditions of it there is polyclonal human IgG or dynamic when being applied to When thing or the mankind, the amino acid residue participates in forming two comprising with the peptide of the CH areas identical amino acid sequence of people Ig with other Sulfide linkage covalently or stablizes key between non-covalent heavy chain, the wherein nucleosides in the VH areas of the selected antigen-specific antibodies of the coding The nucleotide sequence in the CH areas of acid sequence and the coding Ig is operatively coupled together；

Iii) cell expression system for producing the univalent antibody is provided；

Iv) institute is produced by be co-expressed in the cell of the cell expression system of (iii) (i) and the nucleic acid construct of (ii) State univalent antibody.

The antibody of 65. 63, the wherein univalent antibody include

The variable region of the antibody of (i) item 1-26 or the antigen-binding portion thereof in the area, and

(ii) immunoglobulin (Ig) C_HArea or it include C_H2 and C_HThe fragment in 3rd area, the wherein C_HArea or its fragment are modified, So that equivalent to the region of hinge area, and, if immunoglobulin (Ig) is not IgG4 hypotypes, C_HOther regions in area, for example C_H3rd area, do not contain it is any can be with identical C in the presence of polyclonal human IgG_HArea formed disulfide bond or with identical C_HArea Form other covalently or stablize the amino acid residue of key between non-covalent heavy chain.

The antibody of any one of 66. 63-65, wherein heavy chain is modified so that whole hinge is deleted.

A kind of 67. bispecific molecules, including the antibody and the second binding specificity of any one of item 1-62, such as it is right The binding specificity of people's effector cell, people Fc acceptors or φt cell receptor.

A kind of 68. expression vectors, including coding is selected from SEQ ID NO:1-SEQ ID NO:112 one or more amino The nucleotide sequence of acid sequence.

69. according to the expression vector of item 68, further include the constant region of light chain of encoding human antibody, CH or The nucleotide sequence of the constant region of light chain and heavy chains.

A kind of 70. restructuring eucaryons or prokaryotic host cell, it was produced such as resisting that any one of item 1-59 or 61-66 are limited Body.

A kind of 71. pharmaceutical compositions, it includes the antibody limited such as any one of item 1-54, or as item 67 is limited Bispecific molecule, and pharmaceutically acceptable carrier.

Antibody that 72. such as any one of item 1-66 are limited or such as the bispecific molecule defined in item 67, it is used to make For the purposes of medicine.

The antibody of 73. 72 or bispecific molecule, the wherein purposes are for treating cancer.

The antibody of 74. 73 or bispecific molecule, the wherein cancer are selected from the group：Central nerve neuroma, neck Cancer, lung cancer, breast cancer, cancer of the esophagus, cancer of the stomach, liver and courage cancer, cancer of pancreas, colorectal cancer, carcinoma of urinary bladder, kidney, prostate cancer, son The not clear tumour of endometrial carcinoma, oophoroma, chromoma, sarcoma, original origin, bone marrow cancer, ALL, Chronic lymphocytic leukemia and NHL, cutaneum carcinoma, glioma, the cancer of brain, the cancer in uterus and straight The cancer of intestines.

The antibody of 75. 72 or bispecific molecule, the wherein purposes are for treating cancer of pancreas.

The antibody of 76. 72 or bispecific molecule, the wherein purposes are for treating colorectal cancer.

The antibody of 77. 72 or bispecific molecule, the wherein purposes are for treating oophoroma.

The antibody of 78. 72 or bispecific molecule, the wherein purposes are for treating breast cancer.

The antibody of 79. 72 or bispecific molecule, the wherein purposes are for treating prostate cancer.

The antibody of 80. 72 or bispecific molecule, the wherein purposes are for treating carcinoma of urinary bladder.

The antibody of 81. 72 or bispecific molecule, the wherein medicine are used for one or more other therapeutic agent as changed Treat agent treatment of cancer with combinations.

The antibody of any one of 82. 1-66 or the bispecific molecule of item 697 manufacture for treating cancer medicine In purposes.

The purposes of 83. 82, it includes the supplementary features of any one of 74-81 in item.

A kind of 84. methods of the growth and/or propagation for suppressing the tumour cell of the expression tissue factor, including to have need The individual antibody for applying any one of item 1-66 wanted or the bispecific molecule limited such as item 67.

A kind of 85. methods for producing the antibody of any one of item 1-59 or 61-66, methods described includes following step Suddenly：

A) host cell of item 70 is cultivated, and

B) antibody purification from culture medium.

86. a kind of composition for diagnosis, comprising the antibody that such as any one of item 1-67 is limited.

A kind of 87. methods for the presence of tissue factor in detection sample, including：

- make sample allow the antibody or double special with the bispecific molecule of the antibody of item any one of 1-66 or item 67 Property molecule between tissue factor formed complex under conditions of contact；With

- analyse whether to define complex.

A kind of 88. kits for the presence of tissue factor in detection sample, including

The antibody of any one of-item 1-66 or the bispecific molecule of item 67；With

- using the explanation of the kit.

Brief description

Fig. 1：The sequence alignment of antibody of the present invention.SEQ ID NO are given in the bracket on the right of sequence.

It is highlighted according to CDR1, CDR2 and CDR3 of IMGT：The sequence of italic represents CDR1 areas, underlined sequence Row represent CDR2 areas, and the sequence of runic represents CDR3 areas.

Fig. 2：IgG4 sequences (SEQ ID NO:113-114)

SEQ ID NO:113:The amino acid sequence in wild type human IgG4CH area.The sequence of italic represents CH1 areas, highlighted Sequence represents hinge area, and sequence of rules represents CH2 areas, and underlined sequence represents CH3 areas.

SEQ ID NO:114:The amino acid sequence in the hinge-less CH areas of human IgG 4.

Fig. 3：The combination of anti-TF people's monoclonal antibody and TF extracellular domains.Anti- TF people's monoclonal antibody is determined by ELISA to combine in ELISA In combination to the extracellular domain of TF.

Fig. 4：The combination of the TF of anti-TF people's monoclonal antibody and film combination.Anti- TF people's monoclonal antibody is to film combination TF on MDA-MD-231 Combination.With reference to being determined by facs analysis.

Fig. 5：FVIIa is suppressed to be combined with TF.Suppression of the FVIIa to the combination of TF.By ELISA determine FVIIa combine and Suppression of the TF specific humans monoclonal antibody to the combination.

Fig. 6：Suppress the ERK phosphorylations of FVIIa inductions.The suppression of the ERK phosphorylations of FVIIIa inductions, using Western The marking is analyzed.

Fig. 7：Suppress the IL-8 releases of FVIIa inductions.Suppression to the IL-8 releases of FVIIa inductions.MDA-MB-231 exists Cultivate in serum free medium, add TF specific antibodies and FVIIa.The IL-8 of FVIIa inductions is measured by ELISA.

Fig. 8：FXa is suppressed to produce.The suppression that FXa is generated.Test TF specific humans monoclonal antibody suppresses the ability that FXa is generated, The conversion of lower FX to the FXa of TF/FVIIa effects is measured in measurement system using colorimetric FXa specific substrate.

Fig. 9：Suppress blood clotting.Coagulative inhibitors.TF- people is measured with the measurement system in the clotting time for determining TF inductions Suppression of the monoclonal antibody to hemoglutination.

Figure 10：TF- people's monoclonal antibody induces the dissolving of Bx-PC3 cells by ADCC.The ADCC of TF- people's monoclonal antibody induction is to Bx-PC3 The cracking of cell.

Figure 11：Complement component C3c and C4c are deposited on target cell.

Figure 12：The immunohistochemical analysis that TF people's monoclonal antibody is combined with glomerulus.TF- people's monoclonal antibody is exempted to the combination of normal person's kidney Epidemic disease tissue chemical analysis.

Figure 13：The immunohistochemical analysis that TF- people's monoclonal antibody is combined with pancreatic neoplasm.Combination of TF- people's monoclonal antibody to pancreatic neoplasm Immunohistochemical analysis.

Figure 14：Internal effect of TF- people's monoclonal antibody in the MDA-MB-231 tumor xenogeneic grafts set up.TF- people's list Resist the internal effect in the MDA-MB-231 tumor xenogeneic grafts set up in the mammary fat pad of SCID mice.

Figure 15：By intravenous injection TF specific humans monoclonal antibody 011 rear in machin (cynomolgus monkeys) It is determined that bleeding time.Antibody is applied in the 1st (0mg/kg), 8 (1mg/kg), 15 (10mg/kg) and 22 (100mg/kg) days.Go out The blood time (minute), 1,24 and 120 hour measurement function bleeding time and lose blood upon administration.

Figure 16：Internal effect of TF- people's monoclonal antibody in the preventative BX-PC3 tumor xenogeneic grafts set up. Processed with 400 μ g/ mouse within 0 day, dual factors restructuring measurement variance analysis subsequently carries out Bonferroni post-hoc tests：111 pairs KLH：From the 28th day backward：P ＜ 0.05, from the 32nd day backward：P ＜ 0.01 (upper figure).Processed with 300 μ g/ mouse at the 8th day, Then weekly with the process of 150 μ g/ mouse.Dual factors restructuring measurement variance analysis, subsequently carries out Bonferroni post-hoc tests： 111 couples of KLH：From the 35th day backward：P ＜ 0.05 (figure below).

Figure 17：Reorganization reorganization construct and TF domains.TFmm reorganizes construct, domain containing TFmm (upper figure).TFmm reorganization builds Body, domain containing TFhs (figure below).

Figure 18：The combination of anti-TF antibody and TF reorganization reorganization constructs.Anti- TF people's monoclonal antibody on HEK293F cells to expressing TF reorganize construct combination.It show the knot that the different TF expressed on anti-TF people's monoclonal antibody HEK293F cells reorganize construct Conjunction situation, is determined by FACS.Each group shows the data from a dominant clone.X-axis shows different constructs, mould Intend, TFhs, TFmm, TFhsmm, TFmmhs, TFhs1-41mm, TFhs42-84mm, TFhs85-122mm, TFhs123-137mm, TFhs138-159mm,TFhs160-184mm,TFhs185-225mm,TFhs226-250mm,TFmm1-47hs,TFmm48- 90hs,TFmm91-122hs,TFmm123-137hs,TFmm138-159hs,TFmm160-184hs,TFmm185-225hs, TFmm226-250hs。

Figure 19：The combination of people's monoclonal antibody-TF Fab fragments and TF extracellular domains, ELISA.People's monoclonal antibody-TF Fab fragments are to TF's The combination of extracellular domain, is determined by ELISA.

Figure 20：The combination of people's monoclonal antibody-TF Fab fragments and TF extracellular domains, FACS.People's monoclonal antibody-TF Fab fragments are to cell TF Combination, determined by FACS to BxPC3 cells.

Figure 21：Depend on the bind profile of anti-TF people's monoclonal antibody of the number of expressed TF molecules.The combination of anti-TF people Mab Curve depends on the TF molecular numbers of expression.Anti- TF people's monoclonal antibody is determined to expressing the clone of the TF of varying level by FACS With reference to.

Detailed description of the invention

Definition

Term " tissue factor ", " TF ", " CD142 ", " tissue factor antigen ", " TF antigens " and " CD142 antigens " are at this In be used interchangeably, and unless explicitly stated otherwise, including by the natural expression of cell or thin what is transfected with tissue factor gene Any variant of the human tissue factor of expression, isotype and species homologue on born of the same parents.

Term " immunoglobulin (Ig) " refers to glycoprotein related on a class formation, is made up of two pairs of polypeptide chains, a pair light (L) Low molecular weight chain and a counterweight (H) chain, all four chains can be connected by disulfide bond inside.The structure of immunoglobulin (Ig) is Fully characterized.See such as chapters of Fundamental Immunology the 7th (Paul, W., ed., 2nd ed.Raven Press,N.Y.(1989)).In brief, every heavy chain typically by weight chain variable district (abbreviated herein as V_HOr VH) and heavy chain Constant region is constituted.CH is typically made up of three domains：C_H1,C_H2 and C_H3.Every light chain is typically by light chain Variable region is (abbreviated herein as V_LOr VL) and constant region of light chain composition.Constant region of light chain is typically made up of a domain：C_L。 V_HAnd V_LArea can be further subdivided into super changeability area, and (or hypervariable region, it is in terms of the sequence and/or form of structure qualification ring Can have high changeability), also referred to as complimentarity determining regions (CDRs), between them between be studded with it is more conservative, be referred to as framework region (FR) region.Each V_HAnd V_LTypically it is made up of three CDR and 4 FR, from aminoterminal to c-terminus according to following order Arrangement：FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J.Mol.Biol.196,901- 917(1987)).Typically, the numbering of amino acid residue is according to IMGT., Sequences of Proteins in the region Of Immunological Interest, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (" variable domain residue of numbering identical with such as Kabat or according to Kabat " etc. Phrase, is with reference to this numbering system herein for weight chain variable district or light chain variable district).Using this volume Number system, the actual linear amino acid sequence of peptide may be containing less or more amino acid, corresponding to variable region FR or CDR Shorten or insert.For example, weight chain variable district may be in V_HCDR2 residues 52 below comprising a monamino acid insertion (according to Kabat, residue 52a), and behind heavy chain FR residue 82 comprising it is multiple insertion residues (such as according to Kabat, residue 82a, 82b With 82c etc.).The Kabat numberings of the residue of given antibody can be compiled by the homologous region in the antibody sequence and " standard " Kabat Number sequence is compared and is determined.

In the context of the present invention, term " antibody " (Ab) refers to immunoglobulin molecules, immunoglobulin molecules Fragment, or derivatives thereof, it has the ability that combined with antigentic specificity under typical physiological condition, with reasonable time Half life, for example, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours longer, about 48 hours or longer, about 3rd, (such as induction, promotion, raising enough during 4,5,6,7 or more days etc., or any other related functionally restriction And/or the time of the physiologic response associated with the combination of antibody and antigen is adjusted, and/or be enough to raise effector activity for antibody Time).The binding domain with antigen interaction is contained in the heavy chain of immunoglobulin molecules and the variable region of light chain.Antibody (Abs) Constant region can be with the combination of mediated immunity globulin and host tissue or the factor, including immune various cells (are for example imitated Answer thing cell) and complement system component, such as C1q, the first component in complement activation classical pathway.Anti- TF antibody may be used also Being that (such as PNAS USA are shown in the description of double antibody for bispecific antibody, double antibody (diabody) or similar molecule90(14), 6444-8(1993)).Really, bispecific antibody, double antibody that the present invention is provided etc. is except a part for bind tissue factor Or outside tissue factor FVIIa complexs, can be combined with any suitable target.As it was previously stated, unless another within a context Point out outward or substantially contradictory, here, term " antibody " includes that antibody keeps the fragment with antigentic specificity binding ability. Jing shows that the antigen binding function of antibody can be performed by the fragment of full length antibody.Cover the bonding pad in term " antibody " The example of section includes (i) Fab ' or Fab fragments, and one kind is by V_L,V_H,C_LAnd C_HThe monovalent fragment that 1 domain is constituted, Huo Zheru Univalent antibody described in WO2007059782 (Genmab)；(ii)F(ab')₂Fragment, comprising two by hinge area The divalent fragments thereof of the Fab fragments of disulphide bridges connection；(iii) substantially by V_HAnd C_HThe Fd fragments of 1 domain composition；(iv) substantially by V_LAnd V_HThe Fv fragments of domain composition；(v) dAb fragments (Ward et al., Nature341, 544-546 (1989)), it is substantially By V_HDomain is constituted, also referred to as domain antibodies (Holt et al；Trends Biotechnol.2003Nov；21(11):484- 90)；(vi) camel antibodies (camelid) or nano antibody (nanobodies) (Revets et al；Expert Opin Biol Ther.2005Jan；5(1):111-24), and (vii) detached complimentarity determining regions (CDR).And, although two of Fv fragments Domain, V_LAnd V_H, be by different gene codes, but they can by synthesize joint be coupled together with recombination method, should The joint of synthesis can be such that they manufacture as single protein chain, wherein V_LAnd V_HArea matches that to form monovalent molecule (referred to as single Chain antibody or scFv (scFv), are shown in such as Bird et al., Science242, 423-426 (1988) and Huston et al.,PNAS USA 85,5879-5883(1988)).Clearly indicate unless otherwise noted or in context, these single-chain antibodies It is included in the range of term " antibody ".Although these fragments are normally contained within the meaning of antibody, they generally and The specific characteristic of the present invention is constituted independently of one another, shows different biological properties and effectiveness.In the context of the present invention, It is further discussed these and other useful antibody fragment.It is also understood that unless otherwise noted, otherwise term " antibody " is gone back Including with any of technology provide polyclonal antibody, monoclonal antibody (monoclonal antibody), antibody sample polypeptide, for example chimeric antibody and Humanized antibody, and the fragment (Fab) of the reservation of antibody and antigentic specificity binding ability, the known skill Art such as enzyme process cutting (enzymatic cleavage), peptide symthesis and recombinant technique.Produced antibody can have any Isotype.

" anti-TF antibody " is antibody as above, itself and antigen tissue factor specific bond.

As used herein, term " human antibody " is intended to include having from the variable of human germline immunoglobulin's sequence Area and the antibody of constant region.It not is by the amino acid of human germline immunoglobulin's sequential coding that the human antibody of the present invention can include Residue is (such as by external random or direct mutagenesis, or during gene shuffling or by dashing forward that internal somatic mutation is introduced Become).However, as used herein, term " human antibody " is not intended to include following antibody, wherein dynamic from another kind of lactation The CDR sequence of thing species (, such as mouse) germline is grafted on people's Frame sequence.

In a preferred embodiment, antibody of the invention is detached.As used herein, " detached antibody " Following antibody is intended to refer to, it is substantially free of other (for example, specific bond tissues of the antibody with different antigentic specificities The separation antibody of the factor is substantially free of the antibody of antigen of the specific bond in addition to tissue factor).However, specific bond people's group The separation antibody for knitting epi-position, isotype or the variant of the factor can be with other related antigens, such as from the antigen of other species (such as tissue factor inter-species homologue) has cross reactivity.And, detached antibody can be substantially free of other cells Material and/or chemical agent.In one embodiment of the invention, two or more have different antigen-binding specificities " separation " monoclonal antibody is combined in the composition of good restriction.

When two or more antibody are used in this context, term " with ... competition " or " with ... cross competition " be Refer to, two or more antibody competitions combine TF, such as competition binding TF in the measure described in embodiment hereof 6.For one A little antibody pair, the competition in embodiment 6 in assay method is carried out only a kind of antibody is coated on flat board with another kind During competition just it is observed that, otherwise it is but then not all right.As used herein, term " with ... competition " also attempt to cover these groups Close antibody.

As used herein, term " monoclonal antibody " or " monoclonal antibody combination " are referred to single group of molecules Into antibody molecule prepared product.Monoclonal antibody combination shows the single binding specificity to defined epitope and affine Power.Therefore, term " human monoclonal antibodies " refers to the antibody for showing single binding specificity, and it has from people's racial immunity ball The variable region of protein sequence and constant region.Human monoclonal antibodies can be produced by hybridoma, and hybridoma is comprising thin with immortalization B cell that born of the same parents are merged, obtaining from transgenosis or transchromosomal non-human animal (such as transgenic mice), the animal has bag The genome of heavy chain transgene containing people and chain transgene.

As used herein, in the linguistic context of the combination of antibody and predetermined antigens, term " with reference to " be typically it with Equivalent to K_DAbout 10^-7M is less, and such as about 10^-8M is less, and such as about 10^-9M is less, and about 10^-10M Or it is less, or about 10^-11The affinity of M or less is combined (such as when being made with antigen in the equipment of BIAcore 3000 For part, using antibody as analyte be measured by surface plasma body resonant vibration (SPR) technology when), and with it is predetermined anti- Former binding affinity and the heterogenetic antigen (such as BSA or casein) in addition to predetermined antibody or closely related antigen Binding affinity compare, the K with predetermined antigens_DThan the K with non-specific antigen_DIt is low at least 10 times, for example, at least low 100 times, example It is such as at least low by 1, it is 000 times, for example, at least low by 10, it is 000 times, for example, at least low by 100,000 times.Affinity it is low go out amount depend on it is anti- The K of body_D, therefore as the K of antibody_DWhen very low (antibody high special), the affinity of antigen is less than to heterogenetic antigen The amount of affinity can be at least 10,000 times.

As used herein, term " k_d”(sec^-1) refer to the dissociation rate constant of specific antibodies-antigen interaction.It is described Value is also referred to as k_offValue.

As used herein, term " k_a”(M^-1x sec^-1) refer to the association rate constant of specific antibodies-antigen interaction.

As used herein, term " K_D" (M) refer to the Dissociation equilibrium constant of specific antibodies-antigen interaction.

As used herein, term " K_A”(M^-1) the combination equilibrium constant of specific antibodies-antigen interaction is referred to, and lead to Cross k_aDivided by k_dObtain.

Present invention also offers including the V of embodiment antibody_LArea, V_HThe functional variety of area or one or more CDR it is anti- Body.V used in the linguistic context of anti-TF antibody_L、V_HOr the functional variety of CDR still allows for the antibody and retains antibody of the present invention Affinity/affinity and/or specificity/selective at least considerable part (at least about 50%, 60%, 70%, 80%, 90%, 95% or more), and in some cases, this anti-TF antibody can have the affinity bigger than parental antibody, Selective and/or specificity.

These functional varieties typically retain the notable sequence iden with parental antibody.Percentage between two sequences Homogeneity is function (that is, the % homologys=same position number/total positional number x of the number of the same position that sequence is shared 100), while considering the number of breach and the length of each breach, being introduced for of breach carries out optimal between two sequences The needs of comparison.The determination of sequence alignment and percentage identities between two sequences can be realized with mathematical algorithm, as follows Described in the non-limiting examples of face.

Percentage identities between two nucleotide sequences can (can be in http with GCG software kits:// Www.gcg.com is obtained) in GAP programs determined, using NWSgapdna.CMP matrixes, Gap Weight is 40,50,60, 70 or 80, and Length Weight 1,2,3,4,5 or 6.Two percentage identities between nucleotides or amino acid sequence can be with With E.Meyers and W.Miller, Comput.Appl.Biosci4, 11-17 (1988)) algorithm determined that it is Jing is introduced in ALIGN programs (2.0 editions), and using PAM120 residue table is weighted, and Gap Length Penalty is 12, and Gap Penalty is 4.Additionally, the percentage identities between two amino acid sequences can use Needleman and Wunsch, J.Mol.Biol.48, 444-453 (1970)) and algorithm determined that it is introduced in GCG software kits (can be from http:// Www.gcg.com is obtained) GAP programs in, using the matrixes of Blossum 62 or PAM250 matrixes, Gap Weight is 16,14, 12nd, 10,8,6 or 4, Length Weight is 1,2,3,4,5 or 6.

The sequence of CDR variants can by mainly conservative replacement, example different from the CDR sequence of parent antibody sequence As having at least about 35% in the replacement of variant, about 50% or more, about 60% or more, about 70% or more, it is big About 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more (for example About 65-99%, such as about 96%, 97% or 98%) be that conservative amino acid residue is replaced.

The sequence of CDR variants can by mainly conservative replacement, example different from the CDR sequence of parent antibody sequence Such as at least 10 replacements in variant, for example, at least 9,8,7,6,5,4,3,2 or 1 replacements are that conservative amino acid residue is replaced Change.

In the context of the present invention, conservative replacement can be with the ammonia reflected in one or more in following three form Replacement in base acid classification is defined.

For guarding the amino acid residue replaced classification

Other conservative amino acid residues replace classification

1	A	S	T
				2	D	E
3	N	Q
				4	R	K
5	I	L	M
				6	F	Y	W

Other physics of amino acid residue and function classification

Containing alcohol-based residue	S and T
		Aliphatic residue	I, L, V, and M
Cycloolefin is related to residue	F, H, W, and Y
		Hydrophobic residue	A, C, F, G, H, I, L, M, R, T, V, W, and Y
Negatively charged residue	D and E
		Polar residues	C, D, E, H, K, N, Q, R, S, and T
Positively charged residue	H, K, and R
		Little residue	A, C, D, G, N, P, S, T, and V
Minimum residue	A, G, and S
		Participate in the residue that corner is formed	A, C, D, E, G, H, K, N, Q, R, S, P, and T
Flexible residue	Q, T, K, S, G, P, D, E, and R

More conservative replacement packets include：Val-Leu-isoleucine, phenylalanine-tyrosine, lysine- Arginine, alanine-valine and Asp-Glu.

Can also the use-case such as principle introduced in following documents formulate other amino acid packets：Creighton(1984) Proteins:Structure and Molecular Properties(2d Ed.1993),W.H. Freeman and Company。

In one embodiment of the invention, compared with the CDR of the antibody of embodiment, also substantially protect in variant CDR Hold conservative in terms of hydrophilic (hydropathic)/hydrophilic (hydrophilic) property and residue weight/size (for example, Weight classification (weight class) of sequence, hydropathic score or both are retained at least about 50%, at least about 60%, extremely Few about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, Or more (such as about 65-99%)).For example, conservative residue is replaced and is also possible that or can be alternately, Based on conservative substitutions (strong or weak based weight based strong or weak or based on weight Conservation groups) replacement, this is known in the art.

The similar score that retaining similar residue can also or alternately can be determined by using blast program is entered Row measurement (the BLAST 2.2.8 that for example can be accessed by NCBI, using standard setting BLOSUM62, open breach=11 He Prolongation breach=1).Suitable variant shows at least about 45%, for example, at least about 55%, at least greatly compared with parent's peptide About 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or more (such as about 70- 99%) similitude.

As used herein, " isotype " refer to by weight chain constant area gene encode immunoglobulin class (for example IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM).

Term " epi-position " is the protein determinant for referring to specific antibodies.Epi-position is generally by the surface group structure of molecule Into, such as amino acid or sugared side chain, and generally there is specific Three Dimensions Structure, and specific character.Conformation The difference of epi-position and non-comformational epitope is, in the presence of denaturant, and the former combination disappears, and does not disappear with the combination of the latter Lose.Epi-position can include directly participating in amino acid residue (the also referred to as immunodominance component of epi-position of combination (immunodominant component)), and other directly do not participate in combine amino acid residue, for example can specifically be resisted (in other words, these amino acid residues are located at the footprint of specific antigen binding peptide to the amino acid residue of former binding peptide effectively blocking (footprint) in).

As used herein, if antibody (is such as passed through from what is obtained using the system of human immunoglobulin sequence Immunity carries the transgenic mice of human immunoglobulin gene or by screening human immunoglobulin gene storehouse), and its Middle institute human antibodies V domain sequences are in the sequence of amino acid V domains and by the amino acid sequence of the germ-line immunoglobulin gene code With at least 90%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, or for example, at least 99% similitude, then claim the human antibody " being derived from " specific Germline sequences,.Typically, except heavy chain CDR3, from particular person The human antibody of Germline sequences shows and is less than 20 ammonia compared with the amino acid sequence by the germ-line immunoglobulin gene code The difference of base acid, such as less than the difference of 10 amino acid, such as less than 9,8,7,6 or 5, such as less than 4,3,2 Or the difference of 1 amino acid.

As used herein, term " Developing restraint " (such as cell, such as tumour cell) be intended to be included in it is anti- TF antibody contact when, and not with anti-TF antibody contact identical cell growth compared with, cell growth it is any measurable Reduce, for example, suppression growth of cell culture at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.This cell growth is reduced can be occurred by number of mechanisms, such as effector cell phagocytosis, ADCC, CDC and/or Apoptosis.

Term " bispecific molecule " be intended to include it is any have two kinds difference binding specificities reagent, such as albumen Matter, peptide or albumen or peptide complex.For example, molecule can be with reference to the Fc on (a) cell surface antigen and (b) effector surface Acceptor or with (a) cell surface antigen and (b) effector surface on Fc acceptor interactions.Term " bispecific antibody " is intended to bag Any anti-TF antibody is included, it is bispecific molecule.Term " bispecific antibody " also includes double antibody.Double antibody be divalence, The antibody of bispecific, wherein V_HAnd V_LDomain is expressed in single polypeptide chain, but two can not made in same chain are short to one The joint connection of individual domain pairing, forces whereby these domains to be matched with the complementary domain on another chain, and produces two antigens Binding site is (see such as Holliger, P.et al., PNAS USA90,6444-6448(1993),Poljak,R.J.et al.,Structure 2,1121-1123(1994))。

" the defective antibody of effector function " or " effector function defect antibody " refers to following antibody, its activation one Plant or the ability of various effector mechanism (such as complement activation or Fc receptor bindings) significantly reduces or lack nothing.Therefore, effect Cell mediated cytotoxicity (ADCC) and/or the cell toxicant of Complement Dependent that the mediate antibody of thing functional defect antibody is relied on The ability of effect (CDC) significantly reduces or lacks nothing.One example of this antibody is IgG4.

Term " univalent antibody " mean in the context of the present invention antibody molecule can with reference to single antigen molecule, because This nonantigenic crosslinking ability.

Term " stabilized IgG4 antibody " is referred to and is modified the IgG4 antibody for reducing half molecule exchange (see van der Neut Kolfschoten M et al.(2007)Science 14；317 (5844) and bibliography therein, see also Labrijn et al.(2009)Nature Biotechnology,27,767-771)。

As used herein, term " effector cell " refers to the effective stage for participating in immune response (with immune response Cognitive phase and activation stage it is relative) immunocyte.Immunocyte example includes the cell of marrow or lymphatic origin, for example Lymphocyte (such as B cell and T cell, including cytolytic T lymphocyte (cytolytic T cell) (CTL), kill cell, Natural killer cell, macrophage, monocyte, eosinophil, polymorphonuclear cell, such as neutrophil leucocyte, granulocyte, Mast cell and basicyte.Some effector cells express specific Fc acceptors, and perform specific immunologic function.One In a little embodiments, effector cell is capable of the cell cytotoxicity (ADCC) that induction of antibodies is relied on, such as natural killer cell, ADCC can be induced.For example, the monocyte and macrophage for expressing FcR participates in specific killing target cell and to immune system Other components present antigens, or combined with the cell for presenting antigen.In one embodiment, effector cell can be gulped down Bite target antigen or target cell.Specific expression of the FcR on effector cell can by humoral factor, such as cell factor, Adjusted.For example, it was found that the disturbed plain γ (IFN-γ) of the expression of Fc γ RI and/or G-CSF are raised.It is this to be enhanced Expression can increase cytotoxicity of the cell to target for carrying Fc γ RI.Effector cell can swallow or crack target antigen or Target cell.

As used herein, term " carrier " is intended to refer to that the nucleic acid point of another nucleic acid being attached thereto can be transported Son.A type of carrier is " plasmid ", and it refers to circular double-stranded DNA ring, wherein extra DNA sections can be connected.It is another The carrier of type is viral vector, wherein extra DNA sections may be connected in viral genome.Some carriers can Autonomous replication in their host cell is introduced, (such as bacteria carrier with bacterial origin of replication and additional build lactation are moved Thing carrier).Other carriers (such as non-add build mammalian vector) can be incorporated into after being incorporated in host cell In the genome of host cell, replicate together with host genome whereby.And, some carriers can instruct operable company therewith The expression of the gene for connecing.These carriers are herein referred to as " recombinant expression carrier " (or being referred to as " expression vector ").Typically Ground, useful expression vector is often the form of plasmid in recombinant DNA technology.In this manual, " plasmid " and " carrier " can With used interchangeably because plasmid be most frequently with carrier format.However, the invention is intended to including the table of these other forms Up to carrier, such as viral vector (replication defect type retrovirus, adenovirus, adeno-associated virus), they play work(of equal value Energy.

As used herein, term " recombinant host cell " (or being referred to as " host cell ") is intended to refer to wherein introduce The cell of expression vector.It should be appreciated that these terms are intended to not only indicate specific subject cell, these cells are also indicated Offspring.Because there are some modifications in successive generations because of mutation or ambient influnence, these offsprings may be actual On will not be identical with parental cell, but still be included in the range of terminology used here " host cell ".Restructuring place Chief cell includes such as transfectoma (transfectomas), and such as Chinese hamster ovary celI, HEK293 cells, NS/0 cells and lymph are thin Born of the same parents.

As used herein, term " transfectoma " includes the restructuring eukaryotic host cell of expression antibody, and such as CHO is thin Born of the same parents, NS/0 cells, HEK293 cells, plant cell or fungi, including yeast cells.

Term " nonhuman transgenic animal " refers to following non-human animal, and its genome includes one or more people's weights Chain and/or chain transgene or transfection chromosome (integrate or unconformity is in natural gene group DNA of animal), and being capable of table Up to completely human antibody.For example, transgenic mice can have people's chain transgene, and people's heavy chain transgene or the transfection of people's heavy chain Colour solid, so as to when immunity is carried out with the cell of TF antigens and/or expression TF, mouse can produce the anti-TF antibody of people.People's heavy chain Transgenosis can be incorporated in the chromosomal DNA of mouse, as transgenic mice (such as people's monoclonal antibody mouse such as HCo7 or HCo12 mouse) in like that, or people's heavy chain transgene may remain in outside chromosome, as described in WO02/43478 In transfection chromosome KM mouse like that.These transgenosis and transchromosomic mice (collectively referred to herein as " transgenic mice ") can pass through V-D-J recombinates and isotype conversion produces various isotypes (such as IgG, IgA, IgM, IgD and/or IgE) for given antigen Human monoclonal antibodies.The gene that the such specific antibody of coding can also be passed through to import utilizes transgenic nonhuman animal in generation For the antibody of specific antigen, such as by the way that gene is operatively connected with the gene of expression in animal milk.

" treatment " refers to the therapeutical active compound of the present invention for applying effective dose, for alleviating, mitigating, contain or eradicate (healing) illness or morbid state.

" effective dose " is referred under necessary dosage and time-histories, can effectively obtain the amount for expecting treatment results.Treatment is effective The anti-TF antibody of amount can change with many factors, for example the morbid state of individuality, age, sex and body weight, and Anti- TF antibody is in the individual ability for causing desired response in vivo.Therapeutically effective amount or such amount, wherein antibody or antibody portion The benefit in treatment divided exceedes its any toxicity or illeffects.

" anti-idiotype (anti-idiotypic) " (Id) antibody refers to following antibody, its identification typically with antibody The associated unique determinants of antigen binding site.

Invention further aspect and embodiment

As described above, in the first aspect, the present invention relates to a kind of human antibody, it is combined with human tissue factor.

In one embodiment, when being measured with the assay method described in embodiment 13, antibody with tissue because Apparent affinity (the EC that sub- extracellular domain is combined₅₀) be 3nM or lower, such as 0.50nM or lower, such as 0.35nM or lower, example Such as 0.20nM or lower, such as 0.1nM or lower.

In another embodiment, antibody and the expression tissue factor mammalian cell (be for example encoded tissue because The A431 cells of the construct transfection of son) combine, it is preferable that when being measured with the assay method described in embodiment 14, Its apparent affinity (EC₅₀) it is preferably 10nM or lower, such as 8nM or lower, such as 5nM or lower, such as 2nM or more It is low, such as 1nM or lower, such as 0.5 nM or lower, such as 0.3nM or lower.

In another embodiment, the antibody can A431 it is intracellular induction antibody-dependant cytotoxicity, it is excellent Selection of land, when being measured with the assay method described in embodiment 20, EC₅₀It is worth for 2nM or lower, such as 1nM or lower, example Such as 0.7nM or lower, or 0.3nM or lower, such as 0.20nM or lower, or 0.1nM or lower, or 0.05nM or more It is low.

In another embodiment, when being measured with the method described in embodiment 24, the antibody can effectively press down The growth of the MDA-MB-231 tumours that system has been set up, and/or when being measured with the method described in embodiment 26, can suppress The growth of the BxPC3 tumours set up.

In another embodiment, the antibody can suppress the blood coagulation that tissue factor is induced, it is preferable that when with embodiment When assay method described in 19 is measured, median inhibitory concentration is less than 10nM, e.g., less than e.g., less than 5nM, 2nM, example Such as less than 1nM.

In another embodiment, antibody does not suppress blood coagulation.In one embodiment, compared with natural horizontal, coagulate Blood is suppressed most 30%, such as 25%, such as 20%, such as 15%, such as 10% or such as 5%.

In further embodiment, antibody can suppress FVIIa to be combined with tissue factor, it is preferable that when with embodiment When assay method described in 15 is measured, its maximum suppression value is, more than 80%, to be greater than 90% suppression.

In further embodiment, antibody can suppress the MDA-MB-231 cells IL-8 releases that FVIIa is induced, preferably Ground, when being measured with the assay method described in embodiment 17, maximum suppression value is, more than 40%, to be greater than 50%, It is greater than 60% suppression.

In further embodiment, antibody can suppress TF/FVIIa complexs that FX is transformed into into Fxa, it is preferable that when When being measured with the assay method described in embodiment 18, suppress to be less than 50%, e.g., less than 40%, such as 1-30%'s In the range of.

In further embodiment, antibody includes including with following antibody competition bind tissue factor, the antibody Sequence SEQ ID NO:9 VH areas and comprising sequence SEQ ID NO:65 VL areas.

In further embodiment, the combination of antibody of the present invention and tissue factor is not related to following three residue All：The W that tissue factor is 45, the Y of K or 94 of 46.In further embodiment, the combination is not related to following Arbitrary residue：The W of 45, K or 94 of 46 Y (these coded reference mature Ts F, in Genbank entries NP_001984 Position of equal value is 77,78 and 126).

In another embodiment of antibody of the present invention, antibody and following antibody competition bind tissue factor, this resists Body is included comprising sequence SEQ ID NO:37 VH areas and comprising sequence SEQ ID NO:93 VL areas.

In further embodiment, antibody can suppress the ERK phosphorylations that FVIIa is induced, it is preferable that when with embodiment When assay method described in 16 is measured, its median inhibitory concentration is less than 10nM, e.g., less than e.g., less than 5nM, 2nM.

In further embodiment, antibody can suppress ERK phosphorylations, it is preferable that when with described in embodiment 16 When assay method is measured, its median inhibitory concentration is less than 10nM, e.g., less than e.g., less than 5nM, 2nM, and when with fact It is maximum to the suppression of the IL-8 releases of FVII inductions to be less than 10% when applying the assay method described in example 17 and being measured.

In further embodiment, antibody can induce C3c and C4c depositions, it is preferable that wherein according to embodiment 21 Described in be measured, the antibody can induce C3c and C4c deposition.

In further embodiment, when being measured with ELISA, Fab fragments with such as institute in embodiment 28 The EC50 values that the tissue factor extracellular domain stated is combined are less than 0.1 μ g/mL, such as less than such as less than 0.05 μ g/mL, 0.04 μ g/ mL。

In further embodiment, when being measured with ELISA, Fab fragments with such as institute in embodiment 28 The EC50 values that the tissue factor extracellular domain stated is combined are higher than 1.0 μ g/mL.

In further embodiment, Fab fragments are tied with tissue factor extracellular domain as described in example 28 above The EC50 values of conjunction are less than 10 μ g/mL, such as less than such as less than 1 μ g/mL, 0.5 μ g/mL, or less than 0.2 μ g/mL.

In further embodiment, antibody is combined with human tissue factor but not combined with the rat tissue factor, and phase Than in and people TF combination, and reorganization construct 42-84mm combination reduce, the reorganization construct comprising remove amino acid 42-84 Outside people's TF sequences, and amino acid 42-84 is replaced by mouse sequence, as described in Example 27.

In further embodiment, antibody is combined with human tissue factor but not combined with the rat tissue factor, and phase Than in compared to and people TF combination, and reorganization construct 85-122mm combination reduce, the reorganization construct comprising remove ammonia People's TF sequences outside base acid 85-122, and amino acid 85-122 is replaced by mouse sequence, as described in Example 27.

In further embodiment, antibody is combined with human tissue factor but not combined with the rat tissue factor, and phase Than in compared to and people TF combination, and reorganization construct 123-137mm combination reduce, the reorganization construct comprising remove ammonia People's TF sequences outside base acid 123-137, and amino acid/11 23-137 is replaced by mouse sequence, as described in Example 27.

In further embodiment, antibody is combined with human tissue factor but not combined with the rat tissue factor, and phase Than in compared to and people TF combination, and reorganization construct 185-225mm combination reduce, the reorganization construct comprising remove ammonia People's TF sequences outside base acid 185-225, and amino acid/11 85-225 is replaced by mouse sequence, as described in Example 27.

In further embodiment, antibody is combined with human tissue factor but not combined with the rat tissue factor, and phase Than in compared to and people TF combination, and reorganization construct 226-250mm combination reduce, the reorganization construct comprising remove ammonia People's TF sequences outside base acid 226-250, and amino acid 226-250 is replaced by mouse sequence, as described in Example 27.

In further embodiment, compared to and people TF combination, antibody shows and the construct of the reorganization more than a kind With reference to reduction.In one embodiment, antibody shows and the combination of construct 42-84mm and 85-122mm is reduced.At one In embodiment, antibody shows and the combination of construct 123-137mm and 185-225mm is reduced.In one embodiment, resist Body shows and the combination of construct 123-137mm and 185-225mm and construct 226-250mm is reduced.

In further embodiment, antibody can induce C3c and C4c depositions, it is preferable that according to embodiment 21 When being measured describedly, the antibody can induce C3c and C4c depositions.

In an embodiment of antibody of the present invention, the antibody

- and include comprising sequence SEQ ID NO:9 VH areas and comprising sequence SEQ ID NO:The antibody in 65 VL areas is competing Bind tissue factor is striven, and

- not with include comprising sequence SEQ ID NO:37 VH areas and comprising sequence SEQ ID NO:The antibody in 93 VL areas Competition binding tissue factor.

In further embodiment, antibody includes VH CDR3 areas, and it has

A) sequence as follows

-SEQ ID No:12,

-SEQ ID No:16,

-SEQ ID No:20,

-SEQ ID No:24,

-SEQ ID No:28,

Or

B) variant of any sequence, such as amino acid modified with most 1,2,3,4 or 5, preferred to replace, example Such as guard replace, variant.

In further embodiment, antibody includes thering is such as SEQ ID NO:The VH of sequence or its variant shown in 12 CDR3 areas, the wherein variant are included in the modification of one or more positions in position 2,3,6,9 and 11, it is preferable that wherein should Modification is to replace, it is highly preferred that wherein the replacement is selected from following group：

A) R at position 2 is replaced by K,

B) S at position 3 is replaced by A or T,

C) G at position 6 is replaced by T,

D) L at position 9 is replaced by F, and

E) S at position 11 is replaced by Y.

In another embodiment, antibody includes：

A) comprising the sequence SEQ ID NO of CDR1,2 and 3:10th, 11 and 12 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:66th, 67 and 68 VL areas,

F) variant of arbitrary antibody, wherein the variant has most 1,2 or 3 ammonia preferably in the sequence Base acid modification, more preferably amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes VH, its

In further embodiment, antibody includes VL, its

B) and selected from SEQ ID NO:65,69,73, the 77 VL region sequences with 81 are compared with most 20, such as 15 It is individual, or 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes：

In further embodiment, antibody

- and include comprising sequence SEQ ID NO:37 VH areas and comprising sequence SEQ ID NO:The antibody in 93 VL areas is competing Strive bind tissue factor.

In further embodiment, antibody includes VH CDR3 areas, and it has

A) sequence as follows

-SEQ ID No:8,

-SEQ ID No:52,

Or

B) variant of any sequence, such as it is amino acid modified with most 1,2 or 3, it is preferred to replace, for example guard Replace, variant.

In further embodiment, antibody includes：

A) comprising the sequence SEQ ID NO of CDR1,2 and 3:6th, 7 and 8 VH areas, and comprising the sequence SEQ ID of CDR1,2 and 3 NO:62nd, 63 and 64 VL areas,

B) comprising the sequence SEQ ID NO of CDR1,2 and 3:50th, 51 and 52 VH areas, and comprising sequence SEQ of CDR1,2 and 3 ID NO:106th, 107 and 108 VL areas, or

C) variant of arbitrary antibody, wherein the variant has 1,2 or 3 amino acid preferably in the sequence Modification, more preferably amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes VH, its

In further embodiment, antibody includes VL, its

B) and selected from SEQ ID NO:The 61 VL region sequences with 105 are compared with most 20, such as 15, or 10 It is individual, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes：

In further embodiment, antibody

- not with include comprising sequence SEQ ID NO:9 VH areas and comprising sequence SEQ ID NO:The antibody in 65 VL areas Competition binding tissue factor, and

In further embodiment, antibody includes VH CDR3 areas, and it has

A) sequence as follows

-SEQ ID No:32,

-SEQ ID No:36,

-SEQ ID No:40,

-SEQ ID No:56,

Or

In further embodiment, antibody includes：

D) CDR1,2 and 3 sequence SEQ ID NO is included:54,55 and 56 VH areas, and comprising CDR1,2 and 3 sequences SEQ ID NO:110,111 and 112 VL areas, or

E) variant of arbitrary antibody, wherein the variant has 1,2 or 3 amino acid preferably in the sequence Modification, it is highly preferred that amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes VH, its

In further embodiment, antibody includes VL, its

A) and selected from SEQ ID NO:85,89,93 and 109 VL region sequences have at least 80% homogeneity, for example, at least 90%, at least 95%, or at least 98% or 100% homogeneity, or

B) and selected from SEQ ID NO:85,89, the 93 VL region sequences with 109 are compared with most 20, such as 15, or Person 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes：

In further embodiment, antibody with include comprising sequence SEQ ID NO:41 VH areas and comprising sequence SEQ ID NO:The antibody competition bind tissue factor in 97 VL areas.

In further embodiment, antibody includes VH CDR3 areas, and it has

A) sequence as follows

-SEQ ID No:4,

-SEQ ID No:44,

-SEQ ID No:48,

Or

In further embodiment, antibody includes：

A) comprising the sequence SEQ ID NO of CDR1,2 and 3:2nd, 3 and 4 VH areas, and comprising the sequence SEQ ID of CDR1,2 and 3 NO:58th, 59 and 60 VL areas,

B) comprising the sequence SEQ ID NO of CDR1,2 and 3:42nd, 43 and 44 VH areas, and comprising sequence SEQ of CDR1,2 and 3 ID NO:98th, 99 and 100 VL areas,

C) comprising the sequence SEQ ID NO of CDR1,2 and 3:46th, 47 and 48 VH areas, and comprising sequence SEQ of CDR1,2 and 3 ID NO:102nd, 103 and 104 VL areas, or

D) variant of arbitrary antibody, wherein the variant has 1,2 or 3 amino acid preferably in the sequence Modification, it is highly preferred that amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes VH, its

In further embodiment, antibody includes VL, its

B) and selected from SEQ ID NO:57, the 97 VL region sequences with 101 are compared with most 20, such as 15, or 10, or 5,4,3,2 or 1 amino acid modified, more preferably amino acid substitution, such as conserved amino acid replacement.

In further embodiment, antibody includes：

In further embodiment, when being measured with the method described in embodiment hereof 22, the present invention Antibody is less than 5nM, e.g., less than e.g., less than 3.5nM, 2nM with the affinity of tissue factor.

One group of being combined with tissue factor for particularly interesting antibody of the present invention is characterized in that normal or high affinity and high solution From speed (kd).As proved herein, this antibody-like can show that tumour-specific is combined, and be embodied in them and combine cancerous tissue, But do not combine or less combination health tissues.It is not only restricted to any specific theory, it will be assumed that this group of antibody only can be with The cell good combination of expression high level TF, because only that combining when it is bivalent just efficient.The example bag of these antibody Include antibody as described herein 044,098 and 111.

Therefore, in one embodiment, when being measured with the affinity method described in embodiment hereof 22, this The kd of invention antibody is more than 10^-3sec^-1, and when being measured with the affinity method described in embodiment hereof 22, parent 5nM, e.g., less than e.g., less than 1nM, 0.2nM are less than with joint efforts.

In another embodiment, when being measured with the affinity method described in embodiment hereof 22, this The kd of bright antibody is more than 10^-3sec^-1, and/or when being measured with the affinity method described in embodiment hereof 22, ka is big In 5x10⁴mol^-1sec^-1。

In further embodiment, when being measured with the assay method described in embodiment 23, the antibody with Health tissues do not show combination, are particularly not bound with people's glomerulus, but work as with the measure described in embodiment hereof 23 When method is measured, shows with pancreatic neoplasm and combine.

In further embodiment, when being measured with the method described in embodiment hereof 26, the antibody The growth of BX-PC3 tumours set up can effectively be suppressed.

In another embodiment, antibody of the present invention enters with one or more following property：Inhibit proliferaton, suppression are swollen Knurl blood vessel generation, inducing apoptosis of tumour cell are combined with the tissue factor of alternative splicing.

In further embodiment, the present invention antibody with include following antibody competition bind tissue factor：

In further embodiment, the present invention antibody with have following antibody bind tissue factors on it is identical Epi-position：

In further embodiment, the antibody of the present invention includes：

In further, the present invention relates to a kind of anti-TF antibody of monoclonal, it is included with such as SEQ ID NO: The VH areas of the variant of sequence or any sequence shown in 9,1,5,13,17,21,25,29,33,37,41,45,49 or 53, The variant have most 25 it is amino acid modified, such as 20, such as most 15,14,13,12 or 11 are amino acid modified, Such as 10,9,8,7,6,5,4,3,2 or 1 amino acid modified, for example, delete or insert, preferred to replace, for example, guard and replace.

Such as SEQ ID NO:The variant of the sequence shown in 9,1,5,13,17,21,25,29,33,37,41,45,49 or 53 with Any sequence can have at least 80% homogeneity, for example, at least 85% homogeneity or 90% homogeneity or 95% same Property, such as 96% homogeneity or 97% homogeneity or 98% homogeneity or 99% homogeneity.

In one aspect of the invention, the anti-TF antibody of detached monoclonal includes thering is such as SEQ ID NO:65,57, The VL sequences of the variant of sequence or any sequence shown in 61,69,73,77,81,85,89,93,97,101 or 105, institute State variant amino acid modified with for example most 25, such as 20, such as most 15,14,13,12 or 11 amino acid are repaiied Decorations, such as 10,9,8,7,6,5,4,3,2 or 1 amino acid modified, for example, delete or insert, preferred to replace, for example, guard and replace Change.

Such as SEQ ID NO:The variant of the sequence shown in 65,57,61,69,73,77,81,85,89,93,97,101 or 105 There can be at least 80% homogeneity with any sequence, for example, at least 85% homogeneity or 90% homogeneity or 95% same Property, such as 96% homogeneity or 97% homogeneity or 98% homogeneity or 99% homogeneity.

In another embodiment, antibody includes：

A), the VL areas with the sequence selected from the following group：SEQ ID No:65,57,61,69,73,77,81,85,89, 93,97,101 or 105, and the VH areas with the sequence selected from the following group：SEQ ID No:9,1,5,13,17,21,25,29, 33,37,41,45,49 or 53,

B) the above-mentioned variant of any one, wherein the variant preferably only has the conservative replacement in the sequence.

In a preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 65 and have Such as SEQ ID No:The VH areas of the sequence shown in 9, or variant of any one of this two sequences, the variant

A) amino acid modified with most 25, such as 20, such as most 15,14,13,12 or 11 amino acid are repaiied Decorations, such as 10,9,8,7,6,5,4,3,2 or 1 amino acid modified, for example, delete or insert, preferred to replace, for example, guard and replace Change, or

B) with SEQ ID NO:9 or SEQ ID NO:65 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 57 and tool Just like SEQ ID No:The VH areas of the sequence shown in 1, or variant of any one of two sequences, the variant

B) with SEQ ID NO:1 or SEQ ID NO:57 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 61 and tool Just like SEQ ID No:The VH areas of the sequence shown in 5, or variant of any one of two sequences, the variant

B) with SEQ ID NO:5 or SEQ ID NO:61 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 69 and tool Just like SEQ ID No:The VH areas of the sequence shown in 13, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:13 or SEQ ID NO:69 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 73 and tool Just like SEQ ID No:The VH areas of the sequence shown in 17, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:17 or SEQ ID NO:73 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 77 and tool Just like SEQ ID No:The VH areas of the sequence shown in 21, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:21 or SEQ ID NO:77 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes that one has in SEQ ID No:The VL areas of the sequence shown in 81 Have in SEQ ID No with one:The VH areas of the sequence shown in 25, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:25 or SEQ ID NO:81 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 85 and tool Just like SEQ ID No:The VH areas of the sequence shown in 29, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:29 or SEQ ID NO:85 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 89 and tool Just like SEQ ID No:The VH areas of the sequence shown in 33, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:33 or SEQ ID NO:89 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 93 and tool Just like SEQ ID No:The VH areas of the sequence shown in 37, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:37 or SEQ ID NO:93 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 97 and tool Just like SEQ ID No:The VH areas of the sequence shown in 41, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:41 or SEQ ID NO:97 have respectively at least 80% homogeneity, and for example, at least 85% is same Property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 101 and With such as SEQ ID No:The VH areas of the sequence shown in 45, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:45 or SEQ ID NO:101 have respectively at least 80% homogeneity, and for example, at least 85% is same One property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 105 and With such as SEQ ID No:The VH areas of the sequence shown in 49, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:49 or SEQ ID NO:105 have respectively at least 80% homogeneity, and for example, at least 85% is same One property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

In a further preferred embodiment, antibody includes thering is such as SEQ ID No:The VL areas of the sequence shown in 109 and With such as SEQ ID No:The VH areas of the sequence shown in 53, or variant of any one of two sequences, the variant：

B) with SEQ ID NO:53 or SEQ ID NO:109 have respectively at least 80% homogeneity, and for example, at least 85% is same One property or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity are 99% same Property.

The present invention monoclonal antibody can for example by hybridoma method produce, its first in Kohler et al., Nature 256, in 495 (1975)；Or can be produced by recombinant DNA method.Monoclonal antibody can also be with for example Technology described in following documents is separated from phage antibody library：Clackson et al.,Nature352,624-628 And Marks et al., J.Mol.Biol. (1991)222,581-597(1991).Monoclonal antibody can be from any suitable Source obtains.Thus, for example, monoclonal antibody can be obtained from hybridoma, wherein hybridoma can be from purpose antigen immunity Mouse obtain mouse spleen B cell prepare.The form of purpose antigen can be the cell for for example expressing antigen on the surface, or It is the nucleic acid for encoding purpose antigen.Monoclonal antibody can with from from by immunity people or non-human mammal (such as rat, Rabbit, dog, Primate etc.) antibody expressing cells hybridoma obtain.

In one embodiment, antibody of the invention is human antibody.Can be with for the human monoclonal antibodies of tissue factor Produced with the transgenosis or transchromosomic mice that carry part human immune system rather than mouse system.This transgenosis or transfection color Body mouse includes following mouse, is referred to as people monoclonal antibody mouse (HuMAb) and KM mouse respectively here, and collectively referred to herein as " transgenic mice ".

People monoclonal antibody mouse contains the not rearranged people's weight chain variable of coding and constant chain (μ and γ) and light chain variable and constant The mini seat of human immunoglobulin gene (miniloci) of chain (κ) immunoglobulin sequences, and also with the mutation for targeting, Make endogenous μ and κ chains seat inactivation (Lonberg, N.et al., Nature368,856-859(1994)).Therefore, mouse is right Immunity carries out being shown during response the Mouse IgM or κ expression for reducing, also, people's heavy chain for being introduced into and chain transgene are through class Type changes (class switching) and somatic mutation (somatic mutation) so as to produce the human IgG of high-affinity, κ monoclonal antibodies (Lonberg, N.et al. (1994), above；Summary is shown in Lonberg, N.Handbook of Experimental Pharmacology 113,49-101(1994),Lonberg,N.and Huszar,D., Intern.Rev.Immunol.Vol.1365-93 (1995) and Harding, F.and Lonberg, N.Ann.N.Y.Acad.Sci 764536-546(1995)).The preparation of people monoclonal antibody mouse is documented in the following documents： Taylor,L.et al.,Nucleic Acids Research 20,6287-6295(1992),Chen,J.et al., International Immunology 5,647-656(1993),Tuaillon et al.,J.Immunol.152,2912- 2920(1994),Taylor,L.et al.,International Immunology 6,579-591(1994),Fishwild, D.et al.,Nature Biotechnology 14,845-851(1996).See also US 5,545,806, US 5,569,825, US 5,625,126,US 5,633,425,US 5,789,650,US 5,877,397,US 5,661,016,US 5,814, 318,US 5,874,299,US 5,770,429,US 5,545,807,WO 98/24884,WO 94/25585,WO 93/ 1227, WO 92/22645, WO 92/03918 and WO 01/09187.

There is HCo7 mouse a JKD to interrupt (JKD disruption) (such as in its endogenous light chain (kappa) gene Chen et al.,EMBO J.12, 821-830 (1993) is described), in its endogenous heavy chain genes there is a CMD to interrupt (such as Described in the embodiment 1 of WO 01/14424), and with KCo5 people's kappa chain transgene (such as Fishwild et al., Nature Biotechnology 14, 845-851 (1996) is described) and HCo7 people's heavy chain transgene (such as US 5,770, Described in 429).

There is HCo12 mouse a JKD to interrupt (such as Chen et al., EMBO in its endogenous light chain (kappa) gene J.12, 821-830 (1993) is described), in its endogenous heavy chain genes there is a CMD to interrupt (such as the enforcement of WO 01/14424 Described in example 1), and with KCo5 people's kappa chain transgene (such as Fishwild et al., Nature Biotechnology 14, 845-851 (1996) is described) and HCo12 people's heavy chain transgene (such as the reality of WO 01/14424 Apply described in example 2).

In KM Mouse strains, endogenous mouse kappa light chain gene is by such as Chen et al., EMBO J.12,811-820 (1993) ground homozygosis ground like that interrupts, and endogenous mouse heavy gene is by as described in the embodiment 1 of WO 01/09187 Interrupt on homozygosis ground like that.The Mouse strains carry people kappa chain transgene KCo5, such as a Fishwild et al., Nature Biotechnology 14, 845-851 (1996) is described.The mouse germline also carries one by the sections of chromosome 14 People's heavy chain transchromosome that hCF (SC20) is constituted, as described in WO 02/43478.

The splenocyte of these transgenic mices can be used for producing secretion human monoclonal antibodies according to widely-known technique Hybridoma.The human monoclonal or polyclonal antibody of the present invention, or can also lead to from the antibody of the present invention of other species Cross transgenosis mode, by generation with another kind of non-human animal of targeted immune immunoglobulin heavy chain and sequence of light chain transgenosis or Plant, and produce antibody to produce in callable form.Produce with regard to the transgenosis in mammal body, antibody can be on mountain Produce in the milk of sheep, ox and other mammals and from its recovery.See such as US 5,827,690, US 5,756,687, US 5,750,172 and US 5,741,957.

Further, human antibody of the invention or the antibody of the present invention from other species can use it is well known in the art that Technology by display type technology (including but not limited to, phage display, displaying of retrovirus, ribosomal display and other Technology) produced, and further extra maturation can be carried out to gained molecule, such as affine maturation, these skills Art is well-known in the art (see such as Hoogenboom et al., J.Mol.Biol.227, 381 (1991) (bacteriophages Show), Vaughan et al., Nature Biotech14, 309 (1996) (phage displays), Hanes and Plucthau,PNAS USA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene73, 305-318 (1988) (phage display), Scott TIBS17,241-245(1992),Cwirla et al.,PNAS USA87,6378-6382(1990),Russel et al.,Nucl.Acids Research 21,1081-1085(1993), Hogenboom et al.,Immunol.Reviews 130,43-68(1992),Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and US 5,733,743).If producing the antibody in inhuman source using display technique, this A little antibody can be by humanization.

Antibody of the present invention can be any isotype.Desired effector function, example are typically deferred in the selection of isotype Such as ADCC inductions.The example of isotype has IgG1, IgG2, IgG3, and IgG4.Can use people's constant region of light chain, κ or λ, appoint One.If so desired, the type of anti-TF antibody of the invention can be changed by known method.For example, it is originally used for IgM's The IgG antibody that antibody of the present invention can be invented by type conversion cost.Further, type switch technology can be used for one IgG subclass is transformed into another IgG subclass, for example, be transformed into IgG2 from IgG1.Therefore, the effector function of antibody of the present invention can Such as IgG1 is changed over to change by isotype, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody, for Different therapeutical uses.In one embodiment, antibody of the present invention is IgG1 antibody, such as IgG1, κ.

In one embodiment, antibody of the invention is full length antibody, preferably IgG1 antibody, particularly IgG1, and κ resists Body.In another embodiment, antibody of the present invention is antibody fragment or single-chain antibody.

Antibody fragment can be obtained for example with routine techniques by fragmentation (fragmentation), and with herein The fragment that identical mode described in complete antibody is obtained based on purposes screening.For example, F (ab')₂Fragment can be by using stomach egg White enzyme (pepsin) processes antibody and produces.The F (ab') of gained₂Fragment can be acted upon reducing disulphide bridges, produce Fab' pieces Section.Fab fragments can be by being obtained with Papain ferment treatment IgG antibody；Fab' fragments can use pepsin digestion IgG Antibody is obtaining.F (ab') fragment can also be produced by the following Fab' of thioether bond or disulfide-bonded.Fab' fragments are By cutting off F (ab')₂A disulfide bond in hinge area and the antibody fragment that obtains.Fab' segments-segments can be by with also Former agent, such as dithiothreitol (DTT), process F (ab')₂Fragment is obtaining.Antibody fragment can also be by expressing in recombinant cell Encode the nucleic acid of these fragments to produce (see such as Evans et al., J.Immunol.Meth.184,123-38(1995))。 For example, F (ab') is encoded₂The mosaic gene of a fragment part can include：Coding H chain C_H1 domain and the DNA sequence dna of hinge area, with It is afterwards translation termination codon to produce the antibody fragment molecule of this truncation.

In one embodiment, anti-TF antibody is univalent antibody, preferably such as WO2007059782 (Genmab) (this Text is incorporated to by carrying stating) described in univalent antibody, it has in hinge area deletes.Therefore, in one embodiment, antibody is Univalent antibody, wherein such anti-TF antibody including following method by building：

I) nucleic acid construct of the coding univalent antibody light chain is provided, the construct is special comprising the selected antigen of coding The nucleotide sequence in the VL areas of the anti-TF antibody of the opposite sex and the nucleotide sequence in the constant CL areas of coding Ig, wherein the coding is selected The nucleotide sequence in CL areas of nucleotide sequence and the coding Ig in VL areas of antigen-specific antibodies be operably connected Together, and wherein, in the case of IgG1 hypotypes, the nucleotide sequence in CL areas is encoded through modification so that CL areas do not contain There is any amino acid as described below：It is described under conditions of it there is polyclonal human IgG or when animals or humans is applied to Amino acid can form disulfide bond or covalent bond with other comprising with the peptide of the CL areas identical amino acid sequence；

Ii the nucleic acid construct of the coding univalent antibody heavy chain) is provided, the construct includes the selected antigen of coding The nucleotide sequence in the VH areas of specific antibody and the nucleotide sequence in the constant CH areas of encoding human Ig, wherein the core in coding CH areas Nucleotide sequence is through modification so that corresponding to the region of hinge area, and as required by Ig hypotypes, other regions in CH areas, Such as CH3 areas, any amino acid residue as described below is not contained：Under conditions of it there is polyclonal human IgG or when being applied to During animals or humans, the amino acid residue is participated in being included with other and formed with the peptide of people Ig CH areas identical amino acid sequence Disulfide bond covalently or stablizes key between non-covalent heavy chain, the wherein core in the VH areas of the selected antigen-specific antibodies of the coding The nucleotide sequence in the CH areas of nucleotide sequence and the coding Ig is operatively coupled together；

Iii) cell expression system for producing the univalent antibody is provided；

Similarly, in one embodiment, anti-TF antibody is univalent antibody, and it includes：

The variable region of (i) antibody of the present invention as described herein or the antigen-binding portion thereof in the area, and

(ii) immunoglobulin (Ig) C_HArea or it include C_H2 and C_HThe fragment in 3rd area, the wherein C_HArea or its fragment are modified, from And the region equivalent to hinge area is made, and, if immunoglobulin (Ig) is not IgG4 hypotypes, C_HOther regions in area, such as C_H3 Area, do not contain it is any can be with identical C in the presence of polyclonal human IgG_HArea forms disulfide bond or other are covalently or stable The amino acid of key between non-covalent heavy chain.

In further embodiment, the heavy chain of univalent anti-TF antibody is modified so that whole hinge is deleted.

In further embodiment, the antibody is IgG4 hypotypes (see SEQ ID NO:114, SEQ ID NO:113 Hingeless chain variants), but C_H3rd area are modified, so as to manufacture one or more following amino acid substitutions：The Thr of 234 (T) replaced by Ala (A)；The Leu (L) of 236 is replaced by Ala (A)；The Leu (L) of 236 is replaced by Val (V)；273 Phe (F) is replaced by Ala (A)；The Phe (F) of 273 is replaced by Leu (L)；The Tyr (Y) of 275 is replaced by Ala (A).

In another further embodiment, the sequence of the univalent antibody is modified, so as to its do not include it is any N- connects glycosylated acceptor site.

The anti-TF antibody of the present invention also includes single-chain antibody.Single-chain antibody is that following peptide, wherein heavy chain and light chain Fv are connected Pick up and.In one embodiment, the invention provides a scFv (scFv), wherein in single chain polypeptide, the present invention is anti- The peptide linker (typically about 10,12,15 or more amino acid residues) of the heavy chain and light chain flexibility in TF antibody Fv is in list Couple together in one peptide chain.The method of this antibody-like is produced as described in such as following documents：US 4,946,778,Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.Springer-Verlag,New York,pp.269-315(1994),Bird et al.,Science 242,423-426 (1988),Huston et al.,PNAS USA 85,5879-5883(1988)and McCafferty et al.,Nature348,552-554(1990).Single-chain antibody can be univalent, if only using single V_HAnd V_LIf, can be bivalent, If using two V_HAnd V_LIf, or multivalence, if used over two V_HAnd V_LIf.

In one embodiment, anti-TF antibody of the invention is effector function defect antibody.When antibody is used for by resistance The inhibitory action of disconnected TF and during stimulating immune system, this antibody-like is useful especially.For this kind of application, antibody does not have effect Thing function (such as ADCC) is probably favourable, because such function may result in undesirable cytotoxicity.

In one embodiment, the anti-TF antibody of effector function defect is a kind of stabilized IgG4 antibody.Suitably The example for stabilizing IgG4 antibody is following antibody, and wherein 4 CH4 of human IgG 09 (is indicated with EU indexes, seen Kabat et al) arginine by lysine, threonine, methionine or leucine replace, preferably replaced by lysine (as described in WO2006033386 (Kirin)) and/or wherein hinge area include Cys-Pro-Pro-Cys sequences.

In further embodiment, the anti-TF antibody of stabilized IgG4 is the IgG4 antibody for including heavy chain and light chain, Wherein described heavy chain include the constant region of human IgG 4, its corresponding to 409 position at have be selected from Lys, Ala, Thr, Met and The residue of Leu, and/or have at the position corresponding to 405 and be selected from Ala, the residue of Val, Gly, Ile and Leu, and wherein The antibody optionally includes that one or more are replaced, delete and/or insert, but is not not include Cys- in hinge area Pro-Pro-Cys sequences.Preferably, the antibody includes Lys or Ala residues at the position corresponding to 409, or described anti- Replaced by the CH3 areas of human IgG1, human IgG2 or human IgG 3 in Ti CH3 areas.

In further embodiment, the anti-TF antibody of stabilized IgG4 is to include that heavy chain and the IgG4 of light chain resist Body, wherein the heavy chain includes the constant region of human IgG 4, it has selected from Lys, Ala, Thr, Met at the position corresponding to 409 With the residue of Leu, and/or corresponding to 405 position at have be selected from Ala, the residue of Val, Gly, Ile and Leu, and its Described in antibody optionally include one or more other replace, delete and/or insert, and wherein described antibody is in hinge area Including Cys-Pro-Pro-Cys sequences.Preferably, the antibody includes Lys or Ala residues at the position corresponding to 409, or Replaced by the CH3 areas of human IgG1, human IgG2 or human IgG 3 in the CH3 areas of person's antibody.

In further embodiment, the anti-TF antibody of effector function defect is the antibody of non-IgG4 types, for example IgG1, IgG2 or IgG3, it passes through mutation, so as to the ability for mediating effector function (such as ADCC) is lowered or or even disappears Remove.These mutation are in such as Dall'Acqua WF et al., J Immunol.177(2):1129-1138 (2006) and Hezareh M,J Virol.；75(24):It is on the books in 12161-12168 (2001).

In further embodiment, antibody and another moiety of the present invention, are put such as cytotoxic moieties Injectivity isotope or medicine.This antibody can be by by (such as anti-with anti-TF antibody or its fragment by another part TF heavy chain of antibody, L chains or its anti-TF be special/selective fragment) N-terminal or C-terminal chemical coupling produced (see such as Osamu The Antibody Engineering Handbook that Kanemitsu is edited, by Chijin Shokan (1994) are published).It is this Coupled antibody derivative can also be produced by being coupled at suitable internal residues or glycosyl).

Usually, anti-TF antibody described here can by comprising any suitable number of this kind of modified amino acid and/ Or be associated and modified with this kind of coupling substituent.Adaptability under this linguistic context, it is however generally that depending at least substantially The related TF selectivity of the upper anti-TF antibody of parent for retaining underivatized and/or the ability of specificity.For example, at following aspects, It is probably favourable comprising one or more modifications amino acid：Increase polypeptide serum half-life, reduce polypeptide antigen, Huo Zhezeng Plus polypeptide storage stability.For example, common translation modification or posttranslational modification (example are carried out to amino acid during restructuring is produced N connection glycosylations during expressing such as in mammalian cell at N-X-S/T motifs), or modified by synthetic method. The non-limiting examples of modified amino acid include glycosylated amino acid, sulfated amino acids, isoprenylation (such as farnesyl Change, Mang ox base (geranylgeranylated)) amino acid, acetylated amino acids, acylated amino acid, PEGization amino Acid, biotinylation amino acid, carboxylated amino acid, phosphorylated amino acid etc..Be enough to instruct amino acid modified art personnel Bibliography exist in a large number in whole document domain.Code example can be in Walker (1998) Protein Protocols On Cd-Rom, Humana Press, Towata, find in NJ.Modified amino acid can be selected for example from following amino acid Go out：Glycosylated amino acid, PEGization amino acid, farnesylated amino acid, acetylated amino acids, biotinylation amino acid and lipid Amino acid or the amino acid with organic derivative agent coupling that group is coupled.

Anti- TF antibody can also be by being chemically modified, for example to increase its circulation half with polymer covalent coupling Decline the phase.The example of polymer and the method that they are attached on peptide has illustration in such as following documents：US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546.Other examples of polymer include polyoxyethylated polyols With polyethylene glycol (PEG) (such as molecular weight is of about 1,000- about 40,000 PEG, such as about 2,000- about 20, 000, such as about 3,000-12,000g/mol).

In one embodiment, the invention provides anti-TF antibody, itself and the second molecule coupling labeled selected from below： Radionuclide, enzyme, zymolyte, co-factor, fluorescent marker, chemical luminescent marker, peptide tag or magnetic-particle.At one In embodiment, anti-TF antibody can be with one or more antibody fragments, nucleic acid (oligonucleotides), nuclease, hormone, immunity Conditioning agent, chelating agent, boron compound, optical active matter (photoactive agent), dyestuff etc. are coupled.These and other examination Agent can directly or indirectly with anti-TF antibody couplings of the invention.One example of directly coupled second reagent is by sept Part is being coupled.These septs can be insoluble or solvable (see such as Diener et al., Science231, 148 (1986)), and sept can be selected to allow medicament in target site and/or under given conditions from anti-TF antibody release Put.Agglutinin and fluorescent peptide can be included with other example agents of anti-TF antibody couplings.

In one embodiment, there is provided including the anti-TF antibody of one or more radiolabeled amino acids.Radiation Property mark anti-TF antibody can be used for diagnosis and therapeutic purposes (being another possible feature with radiolabeled molecule coupling labeled). Example for the non-limiting label of polypeptide include but not limited to, 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re.Method for preparing radiolabeled amino acids and related peptide derivatives is known in the art (see example Such as Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US 5,697,902.For example, radioactivity is same Position element can be coupled by chloramine-t method.

In one embodiment, anti-TF antibody of the invention includes nucleic acid or the nucleic acid correlation molecule being coupled.At this In bright this aspect, the nucleic acid of coupling is cytotoxicity ribalgilase.In one embodiment, the nucleic acid of coupling is anti- (for example S100A10 targets antisense molecule to phosphorothioate odn, and it can also be in the joint composition or drug combination method of the present invention A kind of independent component, is shown in such as Zhang et al., J Biol Chem.279(3),2053-62(2004)).In an enforcement In scheme, the nucleic acid of coupling is inhibitory RNA molecules (such as siRNA molecule).In one embodiment, the nucleic acid of coupling is Immunostimulatory nucleic acid (such as containing the DNA molecular of immunostimulating CpG motifs).In one embodiment, the core of coupling Acid is expression cassette of the coding schedule up to tumor suppressor gene, anti-cancer vaccine, the inhibiting tumor cell factor or apoptosis agent.These derivatives Can also include being coupled coding schedule up to the nucleic acid of one or more cytotoxic protein (such as plant or bacteriotoxin).

In one embodiment, anti-TF antibody and functional nucleic acid molecule coupling labeled.Functional nucleic acid molecule include antisense molecule, RNA molecule (such as siRNA molecule), fit (aptamer), ribozyme, triplex form molecule (triplex forming ) and external guide sequence (external guide sequence) molecule.Functional nucleic acid molecule can be played with target point The function of the effector of specific activity of son, inhibitor, conditioning agent and stimulant, or functional nucleic acid molecule can have it is independent In the new activity of any other molecule.

In another embodiment, anti-TF antibody of the invention and fit coupling.

In another embodiment, the invention provides a kind of anti-TF antibody, it is coupled with ribozyme.

Any method for anti-TF antibody to be mutually coupled with coupling molecule known in the art, for example as described above , can use, the method being included in described in following documents：Hunter et al.,Nature144,945(1962), David et al.,Biochemistry 13,1014(1974),Pain et al.,J.Immunol.Meth.40,219 And Nygren, J.Histochem.and Cytochem. (1981)30,407(1982).Perhaps eurypalynous cytotoxic compound Can be connected with protein by using the reaction active groups on cytotoxic compound or by using crosslinking agent.Can be with A kind of general reaction active groups for forming stable covalent bond in vivo with amino be isothiocyanate (Means et al., Chemical modifications of proteins(Holden-Day,San Francisco 1971)pp.105-110)。 The group priorities react with the epsilon-amino of lysine.Maleimide is a kind of general reactive group, for cysteine On sulfydryl form stable internal covalent bond (Ji., Methods Enzymol91,580-609(1983)).Monoclonal antibody Covalent bond generally can not be formed with radioactive metal ion, but they can be by using the chelating agent for being covalently attached to antibody And be attached to indirectly on antibody.Chelating agent can by the amido of amino acid residue (Meares et al., Anal.Biochem.142, 68-78 (1984)) and sulfydryl (Koyama, Chem.Abstr.120, 217262t (1994)) and connection, Glycosyl group (Rodwell et al., PNAS USA can also be passed through83,2632-2636(1986),Quadri et al., Nucl.Med.Biol.20, 559-570 (1993)) and connection.It is a kind of because these chelating agents contain two kinds of functional group Bind metal ion, another kind is connected chelating agent with antibody, so they are commonly known as bifunctional chelating agent (Sundberg et al.,Nature 250,587-588(1974))。

In one embodiment, the invention provides a kind of anti-TF antibody, the anti-TF antibody of such as people, itself and therapy section Point, such as cytotoxin, chemotherapeutics, immunodepressant or radio isotope are coupled.This conjugate is referred to herein as " immune conjugate ".It is referred to as " immunotoxin " (immunotoxins) including one or more cytotoxic immune conjugates.

Cytotoxin or cytotoxic agent include any reagent to cell harmful (such as killing cell).For these abilities Goodman et al., Goodman and Gilman's are shown in the medicine of the well-known type in domain and its record of mechanism of action The Pharmacological Basis Of Therapeutics, the 8th edition, Macmillan Publishing Co., 1990.In such as Vitetta, Immunol.Today14, provide and antibody mediated immunity in 252 (1993) and US 5,194,594 Toxin prepares related added technique.

Suitable therapeutic agent for forming immunoconjugates agent of the present invention includes taxol (taxol), Cytochalasin B (cytochalasin B), Gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), emetine (emetine), mitomycin (mitomycin), Etoposide (etoposide), teniposide (tenoposide), Changchun New alkali (vincristine), vincaleukoblastinum (vinblastine), colchicin (colchicin), Doxorubicin (doxorubicin), daunorubicin (daunorubicin), dihydroxy anthracin diketone (dihydroxy anthracin Dione), mitoxantrone (mitoxantrone), plicamycin (mithramycin), actinomycin D (actinomycin D), 1- boldenones (1dehydrotestosterone), glucocorticoid (glucocorticoids), procaine (procaine), totokaine (tetracaine), lidocaine (lidocaine), Propranolol (propranolol) and purine Mycin (puromycin), (such as amethopterin, Ismipur, 6- thioguanines, cytarabine, fluorine are up to drawing for antimetabolite Guest, 5-fluor-uracil, decarbazine (decarbazine), hydroxycarbamide, asparaginase, gemcitabine, Cladribine), alkanisation Agent (such as mustine hydrochlcride, thioepa, Chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), ring phosphinylidyne Amine, busulfan, dibromannitol, Streptozotocin, Dacarbazine (DTIC), procarbazine, mitomycin C, cis-platinum and other Platinum derivatives, such as carboplatin), antibiotic (such as actinomycin D (being previously referred to as D actinomycin D), bleomycin, daunorubicin (being previously referred to as daunomycin), adriamycin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, peace aspergillus Plain (AMC)), diphtheria toxin and correlation molecule (such as diphtheria A chain and its active fragment and hybrid molecule), ricin toxin (such as ricin toxin A or de-glycosylation ricin A streptomycins), cholera toxin, shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk Protease inhibitors, PE, alorin, saponin(e, modeccin, gelatin, abrin A chain, capsule lotus Root toxin A chains, α-broom aspergillin, tung oil tree (Aleurites fordii) albumen, caryophyllin (dianthin), dyers' grapes (Phytolacca Americana) albumen (PAPI, PAPII and PAP-S), balsam pear mortifier, curcin, crotons poison Albumen, Saponaria officinalis (sapaonaria officinalis) mortifier, gelonin (gelonin), mitogellin, limitation are bent Mycin (restrictocin), phenomycin and enomycin toxin.Other suitable coupling molecules include ribalgilase (RNase), deoxyribonuclease (DNase) I, Staphylococcal enterotoxin-A, PAP, diphtheria toxin and Pseudomonad endotoxin.See such as Pastan et al., Cell47, 641 (1986) and Goldenberg, Calif.A Cancer Journal for Clinicians 44,43(1994).Therapeutic agent, its can with as is described elsewhere herein The anti-TF Antibody Combinations of the present invention are applied, and can also be the candidate that can be used for the treatment part of anti-TF antibody couplings of the invention Thing.

In one embodiment, anti-TF antibody of the invention is coupled with chelator linker (such as tiuxetan), the latter Allow antibody coupling in radio isotope.

In further, the present invention relates to a kind of bispecific molecule, it includes sheet as described herein above Anti- TF antibody and the second binding specificity are invented, such as combination to people's effector cell, people Fc acceptors or φt cell receptor is special The opposite sex, or the binding specificity of another epi-position to TF.

Except anti-TF binding specificities and to the binding specificity of people's effector cell, people Fc acceptors or φt cell receptor it Outward, bispecific molecule of the invention may further include the 3rd binding specificity.

The Exemplary bispecific antibodies molecule of the present invention includes (i) two antibody, and one has specificity to TF, separately One specificity having to the second target, both are coupled together, (ii) an independent antibody, and there is one to be specifically directed to for it The chain of TF and one article of special dimolecular second chain, and (iii) of being directed to are with the list for TF and dimolecular specificity Chain antibody.Typically, the second target/the second molecule is the molecule in addition to TF.In one embodiment, the second molecule is cancer Disease antigen/tumor associated antigen, such as carcinomebryonic antigen (CEA), prostate specific antigen (PSA), RAGE (kidney antigen), first tire egg In vain, CAMEL (the CTL- identification antigens in melanoma), CT antigens (such as MAGE-B5 ,-B6 ,-C2 ,-C3, and D；Mage- 12；CT10；NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigen (such as MUC1, mucoprotein- CA125 etc.), gangliosides antigen, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7 and Ep-CAM.In one embodiment, the second molecule is cancer relevant integrin, for example the integrins of 5 β of α 3. In one embodiment, the second molecule is angiogenesis factor or other cancer relevant growth factors, and such as blood vessel endothelium is given birth to The long factor (VEGF), fibroblast growth factor (FGF), EGF (EGF), EGF-R ELISA (EGFR), angiogenin and its acceptor, especially, the acceptor related to cancer progression (such as one of HER1-HER4 acceptors, C-met or RON).Other cancer progression GAP-associated protein GAPs described herein can also be suitable second molecule.

In one embodiment, bispecific antibody of the invention is double antibody.Bispecific antibody also includes crosslinking Or " Heteroconjugate thing " antibody.For example, an antibody in Heteroconjugate thing can be coupled with Avidin, another and biology Element is coupled.This antibody-like has been proposed, for example, by immune system cell target in undesired cell (see such as US 4, 676,980).Heteroconjugate antibodies follow can be prepared with any convenient cross-linking method.

In further, the present invention relates to encode the expression vector of antibody of the present invention.

In one embodiment, expression vector of the invention includes one or more amino acid sequences that coding is selected from the group The nucleotide sequence of row：SEQ ID NO:1-112.

In another specific embodiment, expression vector of the invention includes encoding what one or more were selected from the group The nucleotide sequence of VH amino acid sequences：SEQ ID NO:9,1,5,13,17,21,25,29,33,37,41,45,49 and 53.

In a specific embodiment, the expression vector of the present invention includes one or more choosings that coding is selected from the group From VH CDR3 amino acid sequences nucleotide sequence：SEQ ID NO 4,8,12,16,20,24,28,32,36,40,44, 48,52 and 56.

In another specific embodiment, expression vector of the invention is selected from the group one or more VL including coding The nucleotide sequence of amino acid sequence：SEQ ID NO:65,57,61,69,73,77,81,85,89,93,97,101 and 105.

In another specific embodiment, expression vector of the invention includes one or more that coding is selected from the group The nucleotide sequence of VL CDR3 amino acid sequences：SEQ ID NO:60,64,68,72,76,80,84,88,92,96,100,104 With 108.

In a specific embodiment, the expression vector of the present invention includes encoding one or more above-mentioned amino acid sequences The nucleic acid molecule of the variant of row, the variant have most 25 it is amino acid modified, such as 20, such as most 15,14, 13rd, 12 or 11 it is amino acid modified, such as 10,9,8,7,6,5,4,3,2 or 1 are amino acid modified, for example delete or insert, it is excellent Choosing is replaced, for example, guard and replace, or has at least 80% homogeneity with any sequence, such as with aforementioned arbitrary amino acid Sequence has at least 85% homogeneity or 90% homogeneity or 95% homogeneity, such as 96% homogeneity or 97% homogeneity or 98% homogeneity or 99% homogeneity.

In a further embodiment, expression vector further includes encoding antibody, the light chain of such as human antibody The nucleotide sequence of both constant region, CH or light chain and CH.

This kind of expression vector can be used to recombinate and produce the antibody of the present invention.

Expression vector in the context of the invention can be any suitable carrier, including chromosome, non-chromosome, synthesis Nucleic acid carrier (nucleotide sequence comprising one group of suitable expression control element).The example of this kind of carrier includes spreading out for following carrier It is biological：SV40, bacterial plasmid, phage DNA, baculoviral, yeast plasmid, from plasmid and phage DNA combination carrier, With viral nucleic acid (RNA or DNA) carrier.In one embodiment, encode anti-TF antibody nucleic acid be comprised in naked DNA or In RNA carriers, including such as linear expression element (such as in Sykes and Johnston, Nat Biotech17,355-59 (1997) described in), compact nucleic acid carrier (such as described in US 6,077,835 and/or WO 00/70087), matter Grain carrier such as pBR322, pUC 19/18 or pUC 118/119, " dwarf (midge) " size minimizes nucleic acid carrier (for example In Schakowski et al., Mol Ther3, described in 793-800 (2001)), or as being deposited (precipitated) nucleic acid carrier construct, such as CaP04- precipitation construct (such as in WO 00/46147, Benvenisty and Reshef,PNAS USA 83,9551-55(1986),Wigler et al.,Cell 14,725 , and Coraro and Pearson, Somatic Cell Genetics (1978)7, described in 603 (1981)).This nucleoid Acid vectors and its using being (see such as US 5,589,466 and US 5,973,972) well-known in the art.

In one embodiment, carrier is suitable for expressing anti-TF antibody in bacterial cell.The example bag of this kind of carrier Include expression vector, such as BlueScript (Stratagene), pIN carriers (Van Heeke＆Schuster, J Biol Chem264, 5503-5509 (1989), pET carriers (Novagen, Madison WI) etc.).

Expression vector is also possible that or is selectively suitable for the carrier expressed in Yeast system.It is any to be adapted to Can use in the carrier expressed in Yeast system.Suitable carrier includes, for example, starts containing composition or inducibility (summary is shown in for son, such as the alpha factors, alcohol oxidase and PGH：F.Ausubel et al.,ed.Current Protocols in Molecular Biology,Greene Publishing and Wiley InterScience New York(1987), With Grant et al., Methods in Enzymol153, 516-544 (1987)) carrier.

Nucleic acid and/or carrier can also include the nucleotide sequence of coding secretion/positioning sequence, and secretion/positioning sequence can Polypeptide, such as immature polypeptide chain are targeted in periplasmic space (periplasmic space) or cell culture medium. These sequences are known in the art, and including secretion targeting sequencing or signal peptide, organelle targets sequence (such as nuclear location Sequence, ER stick signals, mitochondrial transport sequence (transit sequence), chloroplast transit sequence), film positioning/grappling Sequence (such as stop-transfer sequence, GPI anchor series) etc..

In the expression vector of the present invention, encode the nucleic acid of anti-TF antibody can include suitable promoter, enhancer and Other be easy to express element or with suitable promoter, enhancer and other be easy to express element be associated.These yuan The example of part include strongly expressed promoter (such as people CMV IE promoters/enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly- (A) terminator sequence, the replication orgin of escherichia coli plasmid product, it is alternatively that label Antibiotic tolerance genes, and/or easily cloning site (such as polylinker).Nucleic acid can also include inducible promoter (technical staff is, it will be recognized that these terms actually describe gene at certain for (relative with constitutive promoter), such as CMV IE Expression under the conditions of a little).

In one embodiment, encoding the expression vector of anti-TF antibody can be placed in or defeated by viral vector In being sent to host cell or host animal.

In an even further aspect, the present invention relates to recombinate eucaryon or prokaryotic host cell, such as transfectoma, its Produce antibody of the present invention as herein defined or as herein defined bispecific molecule of the present invention.The example of host cell Including yeast, bacterium and mammalian cell, such as CHO or HEK cells.For example, in one embodiment, the present invention is provided A kind of cell comprising such nucleic acid, the nucleic acid stability is incorporated in the cellular genome, and comprising for expressing The coded sequence of the anti-TF antibody of the present invention.In another embodiment, the invention provides such cell, it includes non- The nucleic acid of integration, such as plasmid, clay, phasmid or linear expression element, nonconformable nucleic acid is included for expressing this Invent the coded sequence of anti-TF antibody.

In further, the present invention relates to the hybridoma of antibody of the present invention as herein defined can be produced. In even further aspect, the present invention relates to nonhuman transgenic animal, it includes the nucleic acid of encoding human heavy chain and people's light chain, its In the animal or plant can produce the present invention antibody.The generation of this hybridoma and transgenic animals has been described above.

In further, the present invention relates to a kind of method for producing anti-TF antibody of the invention, methods described Comprise the steps：

A) hybridoma of the present invention as described herein above or host cell are cultivated, and

B) antibody of the present invention is purified from culture medium.

In further main aspect, the present invention relates to be used as the as herein defined anti-TF antibody or such as of medicine Bispecific molecule defined herein.

In a still further aspect, the present invention relates to a kind of pharmaceutical composition, it includes：

- anti-TF antibody as herein defined or bispecific molecule as herein defined, and

- pharmaceutically acceptable carrier.

Pharmaceutical composition can be with pharmaceutically acceptable carrier or diluent and any other known adjuvant and figuration Agent is prepared according to routine techniques, such as in Remington:The Science and Practice of Pharmacy, Technology disclosed in 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

Pharmaceutically acceptable carrier or diluent and any other known adjuvant and excipient should be suitable for this Bright selected compound and selected mode of administration.For the carrier of pharmaceutical composition and the applicability of other components with Determine do not have to the desired biological property of selected the compounds of this invention or pharmaceutical composition based on following factor Have significant adverse effect (for example antigen binding is not had substantial effect (such as 10% or lower relative suppression, 5% or Lower relative suppression etc.)).

The pharmaceutical composition of the present invention can also include diluent, filler, salt, buffer, detergent (such as nonionic Detergent, such as polysorbas20 or Tween 80), stabilization agent (such as sugar or without Argine Monohydrochloride), preservative, histologic fixatives, Solubilizer, and/or other be suitable for the material that is included in pharmaceutical composition.

It has been reported that in cancer cell, such as in human colorectal cancer cell, the expression of TF is subject to 2 to drive disease to enter The control of the main transformation event (K-ras oncogene activations and p53 tumor suppressors are inactivated) of journey, its mode depends on MEK/ to have Silk mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) (Yu et al. (2005) Blood 105: 1734)。

The cancer cell of overexpression TF is probably the particularly preferred target of anti-TF antibody of the invention, because each cell can be with With reference to more antibody.Therefore, in one embodiment, the cancer patient of stand-by anti-TF Antybody therapies of the invention is as follows Patient, such as cancer of pancreas, lung cancer or colorectal cancer patients, it has been diagnosed has in the K-Ras of its tumour cell There are one or more mutation in one or more mutation and/or p53.

In an alternate embodiment, the cancer patient of stand-by anti-TF Antybody therapies of the invention is patient, such as pancreas Gland cancer, lung cancer or colorectal cancer patients, it is in K-Ras without mutation.Any specific theory is not only restricted to, some have The tumour cell of K-Ras activation is possible to weaker to the neurological susceptibility of anti-TF Antybody therapies, because the cell being activated in K-Ras In anti-TF antibody affect the effect of Cellular Signaling Transduction Mediated mechanism may be relatively low.

The actual dose level of active component can be changed in pharmaceutical composition of the present invention, can be as special with acquisition Patient, composition or mode of administration are effectively realized the amount of the active component for expecting therapeutic response, and do not have toxicity to patient.Institute The dosage level of choosing depends on multi-medicament kinetic factor, including the work of the particular composition of the present invention or its acid amides for being adopted Property, method of administration, administration time adopts the drainage rate of special compound, the duration for the treatment of, specific with what is adopted Other medicines, compound and/or material that composition is used in combination, age of treated patient, sex, body weight, situation, one As health status and previous medication history, and the well-known similar factor of medical domain.

Pharmaceutical composition can be applied by any suitable approach and pattern.Chemical combination of the present invention is applied in vivo and in vitro The suitable pathways of thing are well-known in the art, it is possible to selected by those skilled in the art.

In one embodiment, pharmaceutical composition of the invention is parenteral administration.

As used herein, term " parenteral administration " and " parenterally applying " are referred to except enteral and local are administered it Outer mode of administration, typically by injection, and including in epithelium, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, socket of the eye, the heart In (subticular) in dirty, in intracutaneous, intraperitoneal, tendon, under transtracheal, subcutaneous, epidermis, joint, under capsule, under arachnoid, vertebra Manage interior, encephalic, intrathoracic, exterior dura and breastbone inner injection and infusion.

In one embodiment, pharmaceutical composition is by intravenous or subcutaneous injection or administered by infusion.

Pharmaceutically acceptable carrier includes any and whole suitably solvents, decentralized medium, coating agent, sterilized and antifungal Agent, isotonic agent, antioxidant and delay absorbent, and the similar reagent with the compounds of this invention PHYSIOLOGICALLY COMPATIBLE.

The suitable aqueous and non-aqueous carrier example that pharmaceutical composition of the present invention can be adopted includes water, salt solution, phosphoric acid Salt buffer salt solution, ethanol, dextrose, polyalcohol (such as glycerine, propane diols, polyethylene glycol etc.), and its suitable mixture, plant Thing oil, such as olive oil, corn oil, peanut oil, cottonseed oil and sesame oil, carboxymethyl cellulose colloidal solution, bassora gum and can The organic ester of injection, such as ethyl oleate, and/or various buffer solutions.Other carriers are that drug world is well-known.

Pharmaceutically acceptable carrier includes aseptic aqueous solution or dispersion and aseptic powdery, for preparing aseptic note immediately Penetrate solution or dispersion.The use of these media and reagent on pharmaceutically active substance is known in the art.Unless any normal Rule medium or reagent are incompatible with reactive compound, and their uses in pharmaceutical composition of the present invention are contemplated herein.

By using coating material, such as lecithin is by the granular size needed for holding in the case of dispersion and logical Cross and use surfactant, suitable mobility can be kept.

The pharmaceutical composition of the present invention can also include pharmaceutically acceptable antioxidant, such as (1) Water-soluble antioxidant Agent, such as ascorbic acid, cysteine hydrochloride, niter cake, sodium pyrosulfite, sodium sulfite and analog；(2) oil-soluble Antioxidant, such as ascorbyl palmitate, butylated hydroxy anisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, nutgall Propyl propionate, alpha-tocopherol, etc.；(3) metal-chelator, such as citric acid, ethylenediamine tetra-acetic acid (EDTA), sorbierite, wine Stone acid, phosphoric acid etc..

The pharmaceutical composition of the present invention can also in the composition include isotonic agent, such as the sugar, polyalcohol in composition, Such as mannitol, sorbierite, glycerine or sodium chloride.

The pharmaceutical composition of the present invention can also for example be prevented containing the adjuvant for being suitable for selected method of administration for one or more Rotten agent, wetting agent, emulsifying agent, dispersant, preservative or buffer, it can improve the preservation life-span of pharmaceutical composition or effective Property.Prepared by the carrier that the compound of the present invention can avoid quick release with protection compound, such carrier such as sustained release agent Type, including implant, transdermal patch and microencapsulation ground induction system.Such carrier can include that gelatin, monostearate are sweet Grease, distearin, biodegradable, biocompatible polymer, such as Ethylene vinyl acetate multipolymer, polyacids Acid anhydride, polyglycolic acid, collagen, poe and PLA, individually or together with wax, or other are well-known in the art Material.Method for preparing this kind of preparation is that those skilled in the art generally know that.See such as Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson are edited, Marcel Dekker, Inc., New York,1978。

In one embodiment, the compound of the present invention can be formulated to guarantee distribution appropriate in vivo.For The pharmaceutical acceptable carrier of parenteral includes that sterile aqueous solution or dispersion are used to prepare sterile injectable solution immediately Or the aseptic powdery of dispersion.It is known in the art that these media and reagent are used for the use of pharmaceutically active substance.It is any normal Rule medium or reagent, as long as it is not incompatible with reactive compound, is contemplated herein them in pharmaceutical composition of the present invention Use.Supplementary reactive compound can also be included in composition.

Pharmaceutical composition for injection typically must be aseptic, and stable under conditions of manufacture and storage.Group Compound can be configured to solution, microemulsion, liposome or other be suitable for the ordered structure of high drug concentration.Carrier can be water Property or non-aqueous solvent or decentralized medium, containing such as water, ethanol, polyalcohol (such as glycerine, propane diols, polyethylene glycol etc.), And its suitable mixture, such as vegetable oil, olive oil, and injectable organic ester, such as ethyl oleate., can for example lead to Cross following means to keep suitable mobility：By using coating, such as lecithin；In the case of a dispersion by keeping Required granular size；And by using surfactant.In many cases it is preferred to ground is in the composition containing isotonic Agent, such as sugar, polyalcohol such as glycerine, mannitol, sorbierite or sodium chloride.The prolongation of Injectable composition absorbs can be passed through In the composition comprising the reagent that can postpone to absorb, such as Monostearate and gelatin, and realize.Can be prepared it is aseptic can Injection solution：The combination that the desired amount of reactive compound is included as needed containing a kind of composition or Multiple components (is for example gone up Cited by face) suitable solvent in, subsequently by sterile micro filter.Usually, dispersion is by by reactive compound Include in the sterile vehicle containing basic dispersion medium and other required compositions (such as from above-named) and prepare. In the case of for the aseptic powdery for preparing sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilized), these processes can produce active component and add any extra powder from the solution for having first passed through aseptic filtration.

The preparation of sterile injectable solution can be by as required, the desired amount of reactive compound being added to one In the suitable solvent of the combination of kind or various compositions cited hereinabove, aseptic microfiltration is subsequently carried out.Usually, the system of dispersion It is standby be by by reactive compound be added to containing basic dispersion medium and it is required other from above-named composition nothing In bacterium medium.In the case of for the aseptic powdery for preparing sterile injectable solution, the example of preparation method is vacuum drying With freeze-drying (lyophilized), these processes are from the previous solution generation active component through aseptic filtration plus any extra phase Hope the powder of composition.

The pharmaceutical composition of the present invention can contain a kind of compound of present invention or the group of various the compounds of this invention Close.

As described above, in one aspect of the method, the present invention relates to the present invention as herein defined for being used as medicine is anti- Body or as here this paper bispecific molecule of the present invention.

The anti-TF antibody of the present invention can be used for various purposes.Especially, antibody of the invention can be used to treat various shapes The cancer of formula.In an aspect, anti-TF monoclonal antibodies of the invention are used to treat various solid cancer types, for example in The tumour of pivot nervous system, head and neck cancer, lung cancer (such as non-small cell lung cancer), breast cancer, cancer of the esophagus, cancer of the stomach, liver and courage cancer (liver and biliary cancer), cancer of pancreas, colorectal cancer, carcinoma of urinary bladder, kidney, prostate cancer, carcinoma of endometrium, The not clear tumour in oophoroma, malignant mela noma, sarcoma (soft tissue, such as bone and muscle), primary source is (namely unknown next Source), leukaemia, Myeloid cancer (such as Huppert's disease), ALL, chronic lymphocytic leukemia and The cancer of NHL, cutaneum carcinoma, glioma, brain, uterus and rectum.

Further, autoimmune inflammation, such as myopathy or multiple sclerosis, can be used as the anti-TF Dan Ke of the present invention The target of grand antibody.

The anti-TF monoclonal antibodies of the present invention can be also used for the treatment of hemostasis (haemostatis).

The related hemostasis illness of cancer can also be used as the target of the intervention of the present invention.

Further, the disease with inflammation, such as myopathy, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, Gout, SpA (spondylarthropathris), ankylosing spondylitis, Reiter syndrome, psoriatic joint Disease, enteropathic arthritis (enterapathric spondylitis), teenager arthropathy (juvenile Arthropathy), reactive arthropathy, infectivity or infection posterior joint inflammation, tuberculous arthritis, viral arthritis, fungi Property arthritis, syphilitic arthritis, glomerulonephritis, latter stage nephropathy, systemic loupus erythematosus, mb.Crohn, ulcerative colitis Inflammation, inflammatory bowel disease, cystic fibrosis, chronic obstructive pulmonary disease (COPD), asthma, allergic asthma, bronchitis, acute gas Guan Yan, chronic bronchitis, idiopathic pulmonary fibrosis or multiple sclerosis, can resist as the anti-TF monoclonals of the present invention The target of body.

The anti-TF monoclonal antibodies of the present invention can be also used for the treatment of hemostasis disease (haemostatis).

In addition, vascular diseases, such as reangiostenosis, myocardial vascular disease, cranial vascular disease, retinopathy (retinopathia) and macular degeneration, include but are not limited to, moist AMD, can be with anti-TF mab treatments.

The anti-TF monoclonal antibodies of the present invention can be also used for treating the patient with cardiovascular danger, such as artery congee Sample hardening, hypertension, diabetes, dyslipidemia and acute coronary syndrome, include but are not limited to, Acute myocardial stalk Extremely, apoplexy.

The anti-TF monoclonal antibodies of the present invention can be also used for inhibition thrombosis, such as DVT, renal embolism, pulmonary embolism, Arterial thrombosis, or for treatment in arterial, peripheral vascular bypass graft or CBG, arteriovenous Shunt, remove arranging thing such as support or Postductal thrombosis.

The anti-TF monoclonal antibodies of the present invention can be also used for suppressing renal ischemic reperfusion injury.

The anti-TF monoclonal antibodies of the present invention can be also used for treating hyperlipoprotememia (hyperlipoproteineimia), hyperparathyroidism (hyperparathyroidism).

It is sick that the anti-TF monoclonal antibodies of the present invention can be also used for treatment vasculitis, ANCA positive vessels inflammation, Behcet.

The anti-TF monoclonal antibodies of the present invention can be also used for blocking the respiratory failure of wound induction, such as acute respiration Distress Syndrome, ALI.

The anti-TF monoclonal antibodies of the present invention can be also used for blocking infection induced organ dysfunction, such as renal failure Exhaust, acute respiratory distress syndrome, ALI.

The anti-TF monoclonal antibodies of the present invention can be also used for treating various thromboembolic disorders, such as by angiopoiesis The thromboembolic disorders that art, myocardial infarction, unstable angina pectoris and coronary artery stenosis cause.

The anti-TF monoclonal antibodies of the present invention can be also used for preventing occasion, for the TF mediations of the sexy dye for the treatment of system Complication, such as septicemia or pneumonia.

The anti-TF monoclonal antibodies of the present invention can be also used for having thrombosis with atherosclerotic blood vessel The preventative process of dangerous patient.

The anti-TF monoclonal antibodies of the present invention can be also used for treating graft versus host disease.

The anti-TF monoclonal antibodies of the present invention can be also used for increasing the implantation of β cellular transplants, prevention in pancreatic islets transplantation Cardiac allograft angiosis (CAV), prevents acute transplant rejection.

The anti-TF monoclonal antibodies of the present invention can be also used for treating has the particulate for being exposed to the loop organization factor The disease of (circulating tissue-factor exposing microparticles), for example but is not limited only to blood vessel Thrombosis, type ii diabetes, AMI, pulmonary hypertension.

Similarly, the present invention relates to be used for suppress expression TF tumour cell growth and/or propagation method, including to The individual antibody or bispecific molecule for applying the present invention of needs.In one embodiment, the tumour cell is related to cancer Disease, such as prostate cancer, lung cancer (such as non-small cell lung cancer), breast cancer, colorectal cancer (such as metastatic colorectum Cancer), cancer of pancreas, carcinoma of endometrium, oophoroma, cutaneous melanoma (cutaneous melanoma), Leukemic Bone Marrow cancer (such as Huppert's disease), ALL, chronic lymphocytic leukemia and NHL, skin The cancer of cancer, prostate cancer, glioma, brain, kidney, uterus, bladder and rectum.

Moreover, it relates to using can with reference to the monoclonal antibody of people TF prepare for treating cancer (for example above One of described concrete cancer idicatio) medicine in purposes.

In one embodiment, it is based in its urine and/or blood with the selection of the patient of anti-TF Antybody therapies The level of tissue factor (TF).In a specific embodiment, the patient to be treated has phase in urine and/or blood To high-caliber TF.For example, the TF levels in the Urine in Patients to be treated can exceed 20ng/ml, such as more than 40ng/ml, example Such as more than 100ng/ml, such as more than 200ng/ml.Alternately, or in addition, the TF levels in patients serum 100pg/ml can be exceeded, such as more than 200pg/ml.This can be determined with such as ELISA.

In a further embodiment of the treatment method of the present invention, the effect to treating during treating is carried out Monitoring, such as in predetermined time point.In one embodiment, measurement urine or the TF levels in blood, example can be passed through Such as effect is monitored by ELISA.In another embodiment, can be determined by being visualized to disease area Effect, visualization is for example by carrying out one or many PET-CT scannings, such as such as of the invention using the anti-TF antibody of mark The anti-TF antibody of mark be scanned.And, the anti-TF antibody of mark, the such as anti-TF antibody of mark of the present invention can be used for Detection produces the tumour of TF, for example, scan to detect using PET-CT.

Dosage in above-mentioned treatment method and application is adjusted, (is for example controlled with the preferable response for providing optimal The property treated response).For example, single bolus (bolus) can be given, the dosage of several segmentations is given within a period of time, or Can according to treatment situation in the urgent need to and proportionally reduce or increasing dosage.Parenteral composi can be formulated into unit Dosage form, in order to being administered and keeping dose uniformity.Unit dosage form as used herein, refer to be adapted as UD (unitary dosage), the physically discrete unit of experimenter to be treated；Each unit contains predetermined Amount reactive compound and required pharmaceutical carrier, the scheduled volume of the reactive compound can be produced through calculating and desired control curative effect Really.The specification of the unit dosage form of the present invention is arranged by following factor and directly determined：The specific characteristic of (a) reactive compound With the work for treating individual sensitivity this to compatibility inherent in concrete result for the treatment to be achieved, and (b) prior art The restriction of property compound.

The effective dose of anti-TF antibody and dosage domain depend on disease or illness to be treated, it is possible to by this area skill Art personnel determine.The non-limiting scope of one example of the therapeutically effective amount of the compounds of this invention is about 0.1-100mg/ Kg, such as about 0.1-50mg/kg, such as about 0.1-20mg/kg, such as 0.1-10mg/kg, such as about 0.5, for example greatly About 0.3, such as about 1, or about 3mg/kg.

There is the doctor or veterinarian of ordinary skill can readily determine that and the pharmaceutical composition needed for prescription for this area Effective dose.For example, for anti-TF antibody contained in pharmaceutical composition, doctor or veterinarian can be from less than realization expectations The desired amount of dosage level of therapeutic effect starts, and gradually increasing dosage, until realizing intended effect.Usually, the present invention The suitable daily dosage of composition is such compound amount, and it can be the lowest dose level for effectively producing therapeutic effect.This Plant effective dose and generally depend on above-mentioned factor.It can be for example intravenous, intramuscular, intraperitoneal or subcutaneous to be administered, for example It is applied near target site.If so desired, can using effective daily dosage of pharmaceutical composition as 2,3,4,5,6 or more Sub-doses, with suitable administration spaced apart, optionally take unit dosage form interior daily.Although the compound of the present invention It is possible to be administered alone, it is preferred that applying compound in the form of above-mentioned pharmaceutical composition.

In one embodiment, anti-TF antibody can be with 10-500mg/m weekly², such as 200-400mg/m²Dosage Administered by infusion.This administration can repeat, such as 1-8 time, such as 3-5 time.Administration can be by lasting 2-24 hours, example Carry out such as the continuous infusion of 2-12 hours.

In one embodiment, anti-TF antibody can delay by within a long period, such as more than 24 hours Slow continuous infusion carrying out, to reduce toxic and side effect.

In one embodiment, anti-TF antibody can use 250mg-2000mg, such as 300mg, 500mg weekly, The dosed administration of 700mg, 1000mg, 1500mg or 2000mg, at most gives 8 times, such as 4-6 time.Administration can be by 2-24 Hour time in, such as continuous infusion is carrying out in the time of 2-12 hours.This dosage regimen can as required repeat 1 It is secondary or multiple, for example it is repeated 1 times behind 6 months or 12 months or repeatedly.Dosage can after measurement administration in blood this The amount of bright compound determining or adjust, for example in the following manner：Take out biological sample and use and target anti-TF antibody of the invention Antiidiotype (anti-idiotypic) antibody of antigen binding domain.

In one embodiment, anti-TF antibody can be administered by maintenance therapy, such as at 6 months or longer Period in 1 times a week.

In one embodiment, anti-TF antibody can be by following administration, including the once infusion present invention's Anti- TF antibody, the of the invention anti-TF antibody that subsequently infusion is coupled with radio isotope.The program can be after such as 7-9 days Repeat.

Used as non-limiting examples, treatment of the invention can be provided as the daily dosage of the compounds of this invention, In an amount of from about daily 0.1-100mg/kg, such as daily 0.5,0.9,1.0,1.1,1.5,2,3,4,5,6,7,8,9,10,11, 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,40,45,50,60,70,80,90 Or 100mg/kg, the 1st after treatment starts, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39, or at least one of 40 days, or At least one of person the 1st, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 week is carried out, or Person its any combinations, using single dose or the dosage per gradation in 24,12,8,6,4 or 2 hours, or its any combination.

" effective dose " for oncotherapy can be being weighed with the ability of its stable disease process.Compound suppresses cancer The ability of disease can be estimated in the animal model system that can be predicted the effect in human tumour.Or, composition this Planting property can check that compound Cell growth inhibition or inducing cell wither by external test method known to technical staff The ability died is estimated.The therapeutic compounds of therapeutically effective amount can reduce tumor size, or otherwise mitigation is received The symptom of examination person.One of ordinary skill in the art can determine the physique of this tittle, i.e. experimenter, receive according to following factor The order of severity of examination person's symptom and selected concrete composition or method of administration.

Anti- TF antibody can also prophylactically be administered, and be sent out with reducing the danger of generation cancer, postponing event in cancer progression Raw beginning, and/or the danger recurred is reduced during cancer mitigates.For by the way that its is internal known to other biological factors The patient that there is tumour but be difficult to positioning tumor is useful especially.

Anti- TF antibody can be to apply in combination treatment, i.e., related with disease to be treated or illness to other treatment Agent is combined.Therefore, in one embodiment, the medicine containing antibody is used for and one or more other therapeutic combination, Such as cytotoxic agent, chemotherapeutics or antiangiogenic agent.

This combined administration simultaneously, respectively or can be carried out sequentially.For being administered simultaneously, reagent can suitably as one Individual composition is applied as other composition.Therefore the present invention additionally provides and is related to expression as above for treatment The method of the disease of TF cells, the method includes applying the present invention with one or more combination with other therapeutic agents as described below Anti- TF antibody.

In one embodiment, the invention provides a kind of for treating the cell for being related to express TF in subject The method of disease, the method includes applying the of the invention anti-TF antibody and at least one of therapeutically effective amount to the experimenter of needs Chemotherapeutics.

In one embodiment, the invention provides for treatment or the method for pre- anti-cancer, the method is included to need The object wanted applies the of the invention anti-TF antibody of therapeutically effective amount and at least one chemotherapeutics.

In one embodiment, the invention provides the anti-TF antibody of the present invention is being prepared for at least one cancer The chemotherapeutics of disease applies together the purposes come in the pharmaceutical composition for the treatment of cancer.

In one embodiment, this chemotherapeutics can be selected from antimetabolite, for example amethopterin, Ismipur, 6- thioguanines, cytarabine, fludarabine, 5 FU 5 fluorouracil, decarbazine, hydroxycarbamide, asparaginase, gemcitabine, Cladribine and similar reagent.

In one embodiment, this chemotherapeutics can be selected from alkylating reagent, such as mustargen, phosphinothioylidynetrisaziridine (thioepa), Chlorambucil, melphalan, card chlorine mustard (BSNU), lomustine (CCNU), endoxan, busulfan, two Bromine mannitol, streptozocin, Dacarbazine (DTIC), procarbazine, mitomycin C, cis-platinum and other platinum derivatives, for example, block Platinum, and similar medicine.

In one embodiment, this chemotherapeutics can be selected from antimitotic agent, such as taxanes (taxanes), such as Docetaxel (docetaxel) and taxol, and vinca alkaloids, such as eldisine, Changchun is new Alkali, vincaleukoblastinum and vinorelbine.

In one embodiment, this chemotherapeutics can be selected from topoisomerase enzyme inhibitor, for example Hycamtin or according to It is vertical to replace health.

In one embodiment, this chemotherapeutics can be selected from Cell growth inhibition medicine, for example, Etoposide and replace Buddhist nun moors glycosides.

In one embodiment, this chemotherapeutics can be selected from growth factor receptor inhibitors, and such as ErbB1 (EGFR) suppresses Agent (such as Iressa, Erbitux (Cetuximab), Erlotinib and similar medicine), ErbB2 (Her2/neu) inhibitor is (for example Trastuzumab and similar medicine) and similar medicine.

In one embodiment, this chemotherapeutics can be selected from tyrosine kinase inhibitor, such as Imatinib (Glivec, Gleevec STI571), Lapatinib, PTK787/ZK222584 and similar medicine.

In one embodiment, the invention provides a kind of be related in subject express the cell of TF for treatment The method of disease, the method includes applying the of the invention anti-TF antibody and at least one of therapeutically effective amount to the experimenter of needs Angiogenesis, neovascularization and/or other vascularization inhibitor.

The example of this angiogenesis inhibitor is urokinase inhibitors, NMPI (such as Ma Li Ma Sita, Neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar medicine), endothelial cell migration and propagation Inhibitor (such as TNP-470, squalamine, methoxyestradiol, combretastatin class (combretastatins), endostatin, Angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) and similar medicine), angiogenesis growth factor antagonist (for example ZD6474, SU6668, the antibody for anti-angiogenesis agent and/or its acceptor (such as VEGF, bFGF, and angiopoietin-1), Thalidomide, thalidomide analogs (such as CC-5013), Sugen 5416, SU5402, anti-angiogenic generation ribozyme are (for example Angiozyme), interferon-' alpha ' (such as Interferon a2a), suramin and similar medicine), VEGF-R kinase inhibitors and other are anti- Blood vessel generation tyrosine kinase inhibitor (such as SU011248), endothelial specificity integrin/survival signaling conduction depressant drug (such as vitaxin and similar medicine), copper antagonist/chelating agent (such as tetrathiomolybdate, captopril and similar medicine), carboxylic Amine triazole (carboxyamido-triazole, CAI), ABT-627, CM101, IL-12 (IL-12), IM862, PNU145156E and suppress blood vessel generation nucleic acid molecule (such as cDNA of his fourth of antisense-VEGF-cDNA, encoding both, The cDNA of the coding p53 and cDNA of coding deficiency vegf receptor -2) and similar medicine.

Other examples of these blood vessel generations, neovascularization and/or other vascularization inhibitor are anti-angiogenic generation heparin Derivative and correlation molecule (such as Heparinase I II (heperinase III)), Temozolomide, NK4, macrophage migration suppress The factor (MIF), COX-2 inhibitors, oxygen deficient induction factor 1, anti-angiogenic generation isoflavones, Oltipraz, aspergillus fumigatus Element and the like, SMS 201-995, pentosane polysulfate ester, tecogalan sodium, DALT, tumor chalone (tumstatin), thrombospondin (thrombospondin), NM-3, combrestatin, canstatin, Ah cutting down he Spit of fland (avastatin), for antibody (such as anti-α-v/beta-3 integrins and anti-of other associated targets Kininostatin monoclonal antibodies) and similar medicine.

In one embodiment, for and for treating the antibody combined treatments for using of the anti-TF of disease as described above Agent can be that antitumor immune is former, such as cancer antigen/tumor associated antigen (such as epithelial cell adhesion molecule (EpCAM/ TACSTD1), MUC-1 (MUC1), carcinomebryonic antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer correlated virus vaccine (such as Human-papilloma Vaccine), the heat shock egg in tumour source White and similar medicine.In many other suitable cancer antigen/tumor associated antigen and known in the art that this paper elsewheres are recorded Similar molecule can also be further or alternately with such an implementation.Antitumor immune originality peptide also includes anti- Idiotype " vaccine ", such as BEC2 anti-idiotypes, mitumomab, CeaVac and related anti-idiotype, for MG7 The anti-idiotype of antibody, and other anticancer anti-idiotypes are (see such as Birebent et al., Vaccine.21 (15),1601-12(2003),Li et al.,Chin Med J(Engl).114(9),962-6(2001),Schmitt et al.,Hybridoma.13(5),389-96(1994),Maloney et al.,Hybridoma.4(3),191-209(1985), Raychardhuri et al.,J Immunol. 137(5),1743-9(1986),Pohl et al.,Int J Cancer.50(6),958-67(1992),Bohlen et al.,Cytokines Mol Ther.2(4), 231-8 (1996) and Maruyama,J Immunol Methods. 264(1-2),121-33(2002)).This anti-idiotype is also with optionally With carrier conjugation, the carrier can be synthesis (typically inert) molecular vehicle, protein (such as keyhole limpet hemocyanin (KLH) (see such as Ochi et al., Eur J Immunol.17(11), 1645-8 (1987)) or cell (for example red blood cell- See such as Wi et al., J Immunol Methods.122(2),227-34(1989))。

In one embodiment, for for treating the antibody combined therapeutic agents for using of anti-TF of illness as described above Can be the inhibiting tumor cell factor, chemotactic factor (CF) or its combination.The example of suitable cell factor and growth factor include IFN γ, IL-2、IL-4、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、IL-18、IL-23、IL-24、IL-27、IL-28a、 IL-28b, IL-29, KGF, IFN α (such as INF α 2b), IFN β, GM-CSF, CD40L, Flt3 part, stem cell factor, Anxi Department's booth and TNF α.Suitable chemotactic factor (CF) includes Glu-Leu-Arg (ELR)-feminine gender chemotactic factor (CF), such as from people CXC and C-C IP-10, MCP-3, the MIG, and SDF-1 α of chemotactic factor (CF) family.Suitable cell factor includes cell factor derivative, cell Factor variant, cytokine fragment and cell factor fusion protein.And/or, these and herein other are related to natural The method or purposes of the nucleic acid of raw encoded peptide can be carried out by " gene activation " and homologous recombination genetic upregulation techniques, such as Described in following documents：US 5,968,502, US 6,063,630 and US 6,187,305 and EP 0505500.

In one embodiment, for can with the antibody combined therapeutic agents for using of anti-TF for treating illness as described above Being cell cycle control/Apoptosis regulator (or " conditioning agent ").Cell cycle control/Apoptosis regulator may include Target and adjust the molecule of cell cycle control/apoptosis regulator, such as (i) cdc-25 (such as NSC 663284), (ii) mistake Degree stimulate the cell cycle cyclin rely on kinases (for example Flavopiridol (flavopiridol) (L868275, ), HMR1275 7- hydroxyls staurosporin (7-hydroxy-staurosporine) (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) Telomerase conditioning agent (such as BIBR1532, SOT-095, GRN163 and in example Composition as described in US 6,440,735 and US 6,713,055).May interfere with apoptotic pathways molecule it is unrestricted Property example include related apoptosis-inducing ligand (the TRAIL)/parts of apoptosis -2 (Apo-2L) of TNF, activate TRAIL acceptors Antibody, IFN and antisense Bcl-2.

In one embodiment, for can be with the antibody combined therapeutic agents for using of anti-TF for treating above-mentioned disease Hormone regulator, such as antiandrogen and the medicine of antiestrogenic therapy.The example of these hormone regulators has tamoxifen Sweet smell, idoxifene, fulvestrant, Droloxifene, Toremifene, Raloxifene, diethylstilbestrol, ethinylestradiol/ethinyloestradiol, anti-hero Hormone (antiandrogene) (such as Drogenil (flutaminde)/Flutamide (eulexin)), progesterone (such as caproic acid hydroxyl Progesterone, Medroxyprogesterone/Progevera, megestrol acetate acetate/megace), adrenocorticotro (such as hydrocortisone, Metacortandracin), Relefact LH-RH (and its analog or other LHRH activators, such as Buserelin and Ge Sherui Woods), aromatase inhibitor (such as Anastrozole/Arimidex, aminoglutethimide/cytraden, Exemestane), hormone inhibitors (such as Octreotide/Sandostatin LAR Depot) and similar medicine.

In one embodiment, the antibody combined therapeutic agents for using of anti-TF for the above-mentioned disease of same treatment can be Anti-allergy lack (anti-anergic) medicine (such as micromolecular compound, protein, glycoprotein or blocking to tumour and The antibody of cancer antigen tolerance).The example of these compounds is can to block the active molecules of CTLA-4, such as MDX-010 (Yi Pu Li Muma (ipilimumab)) (Phan et al., PNAS USA100,8372(2003))。

In one embodiment, the antibody combined therapeutic agents for using of anti-TF for the above-mentioned disease of same treatment can be The replication-defective adenoviral of the nucleic acid containing tumor suppressor gene or carrier, such as encoding human restructuring wild type p53/SCH58500 Deng；Target the antisensenucleic acids of oncogene, mutation or not modulated gene；Target mutation or not modulated gene siRNA.It is swollen The example of knurl suppression target includes such as BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC.

In one embodiment, the antibody combined therapeutic agents for using of anti-TF for the above-mentioned disease of same treatment can be Anticancer nucleic acid, such as genasense (augmerosen/G3139), LY900003 (ISIS 3521), ISIS 2503, OGX- 011 (ISIS 112989), LE-AON/LEraf-AON (the c-raf ASONs/ISIS-5132 of liposome), MG98 and other target PKC α, clusterin (clusterin), IGFBP, PKA, cyclin D1 or Bcl- The antisensenucleic acids of 2h.

In one embodiment, the antibody combined therapeutic agents for using of anti-TF for the above-mentioned disease of same treatment can be Anticancer inhibitory RNA molecules are (see such as Lin et al., Curr Cancer Drug Targets.1(3),241-7 (2001),Erratum in:Curr Cancer Drug Targets.3(3),237(2003),Lima et al.,Cancer Gene Ther.11(5),309-16(2004),Grzmil et al.,Int J Oncol. 4(1),97-105(2004), Collis et al.,Int J Radiat Oncol Biol Phys.57(2Suppl),S144 (2003),Yang et al.,Oncogene.22(36), 5694-701 (2003) and Zhang et al., Biochem Biophys Res Commun.303(4),1169-78(2003))。

The composition and administering drug combinations method of the present invention also includes administration of nucleic acid vaccine, for example, encode these cancer antigens/swollen Knurl related antigen naked DNA vaccine (see such as US 5,589,466, US 5,593,972, US 5,703,057, US 5, 879,687, US 6,235,523, and US 6,387,888).In one embodiment, administering drug combinations method and/or joint group Compound includes autovaccine composition.In one embodiment, combining composition and/or administering drug combinations method includes full cell (dendron of such as fibroblast of expression recombinant il-2, expression recombinant cytokine is thin for vaccine or cytokine-expressing cellular Born of the same parents etc.) (see such as Kowalczyk et al., Acta Biochim Pol.50(3),613-24(2003),Reilly et al.,Methods Mol Med. 69, 233-57 (2002) and Tirapu et al., Curr Gene Ther.2(1),79-89 (2002)).Another example of autogenous cell method of this combined method that can be used for the present invention isIndividuation Immunotherapy method (being previously referred to as GTOP-99) (- Redwood city of Genitope companies, CA, USA).

In one embodiment, the invention provides joint composition and co-administer method, wherein anti-TF antibody with The combinations such as virus, virus protein or common use.For example, replication defective virus, its be generally possible to carry out in vivo 1 wheel or Several wheels are replicated, and target tumour cell, can serve as the component of this composition and method.This kind of virus agent can be with Nucleic acid comprising encoding immune stimulant (such as GM-CSF and/or IL-2), or accompany with it.Natural oncolytic virus and These recombination oncolytic viruses (such as HSV-1 viruses, reovirus, replication defective and duplication responsive type adenovirus etc.) can be used Make the component of these method and compositions.Therefore, in one embodiment, the invention provides joint composition and combine to Prescription method, wherein anti-TF antibody is combined or common use with oncolytic virus.These viral examples include oncolytic adenovirus and blister Exanthema virus, it can be or not be modification virus (see such as Shah et al., J Neurooncol.65(3),203-26 (2003),Stiles et al.,Surgery. 134(2),357-64(2003),Sunarmura et al., Pancreas.28(3),326-9(2004),Teshigahara et al.,J Surg Oncol.85(1),42-7(2004), Varghese et al.,Cancer Gene Ther.9(12),967-78(2002),Wildner et al.,Cancer Res.59(2),410-3(1999),Yamanaka,Int J Oncol.24(4), 919-23 (2004) and Zwiebel et al.,Semin Oncol. 28(4),336-43(2001))。

The joint composition and administering drug combinations method of the present invention can also relate to " full cell " and " adoptive " immunization therapy Method.For example these methods can include infusion or be transfused again immune system cell (such as tumor infiltrating lymphocyte (TIL), Such as CD4⁺And/or CD8⁺T cell (T cell for for example being expanded with tumour specific antigen and/or genetic enhancer), expresses the B of antibody Cell or other antibody produce/and it is in delivery cell, dendritic cells (are for example expressed the restructuring dendritic cells of antibacterial agent, are expanded with DC Increase the dendritic cells of agent such as GM-CSF and/or Flt3-L culture, and/or the dendritic cells of load tumor associated antigen), it is antitumor NK cells, so-called hybrid cell, or its combination.Cell lysate can also be used for these method and compositions.Can be used for these The cell " vaccine " just in clinical testing of aspect includes Canvaxin^TM,APC-8015(Dendreon),HSPPC-96 (Antigenics), andCell lysate.From cancer cell come off (shed from) antigen and its mixture (see such as Bystryn et al., Clinical Cancer Research Vol.7,1882-1887, July 2001), appoints Selection of land mixes with adjuvant such as alum, it is also possible to used as the component in these methods and joint composition.

In one embodiment, anti-TF antibody can be with Inoculation method (internal vaccination Method) combination transfer is to patient.Inoculation refers to the tumour or cancer cell death of the induction in patient's body, and such as medicine is lured The cell death of the tumour cell of induction is led or radiates, it is typically resulted in, and overall for (i) tumour cell or (ii) tumour is thin The part of born of the same parents, including (a) secretory protein, glycoprotein or other products, (b) embrane-associated protein or glycoprotein or other associate with film Or the component being inserted in film, and/or the initiation of the immune response of (c) intracellular protein or other intracellular members.Connect in vivo The immune response for planting induction can be that body fluid type (i.e. antibody-complement mediation) or cell-mediated type (for example can recognize that quilt in vivo The generation and/or increase of the endogenous cell toxic T lymphocyte of the tumour cell of kill or part thereof).Except radiotherapy it Outward, can be used to induce the death of neoplastic cells and the medicine of Inoculation and the non-limiting example of reagent to be conventional chemotherapy Agent, cell cycle inhibitor, anti-angiogenic drug, monoclonal antibody, cell death inducer and signal transduction inhibitor.

Other are that differentiation is lured possibly as the example that therapeutic agent is combined the anticancer to treat above-mentioned disease with anti-TF antibody Lead agent, retinoic acid analog (such as all-trans retinoic acid, 13- cis retinoic acids and similar medicine), novel vitamin D analogues (for example Seocalcitol and similar medicine), the inhibitor of following material：ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta、Flk2、Flt4、FGFR1、FGFR2、FGFR3、FGFR4、TRKA、TRKC、c-met、Ron、Sea、Tie、Tie2、 Eph, Ret, Ros, Alk, LTK, PTK7, and similar medicine.

Other are tissue eggs possibly as the example that therapeutic agent is combined the anticancer to treat above-mentioned disease with anti-TF antibody White enzyme B, the conditioning agent of cathepsin D's dehydrogenase activity, glutathione-S-transferase (such as glutamyl cysteine synthesis Enzyme (glutacylcysteine synthetase) and lactic dehydrogenase), and similar medicine.

Other anticancer examples for being combined to treat above-mentioned disease with anti-TF antibody possibly as therapeutic agent are Estramustines And epirubicin.

Other anticancer examples for being combined to treat above-mentioned disease with anti-TF antibody possibly as therapeutic agent are HSP90 suppressions Preparation, such as 17-AAG, for the antibody of tumour antigen such as PSA, CA125, KSA etc., integrin Such as integrin β 1, VCAM inhibitor and similar medicine.

Other anticancer examples for being combined to treat above-mentioned disease with anti-TF antibody possibly as therapeutic agent are that calcium adjusts phosphoric acid Enzyme (calcineurin) inhibitor (such as valspodar, PSC 833 and other MDR-1 or p- glycoprotein inhibitors), TOR suppressions Preparation (such as sirolimus, everolimus and rapamycin), and " lymphocyte homing " mechanism inhibitor (such as FTY720), With on the cellular signal transduction influential reagent of tool, such as adhesion molecule inhibitors (such as anti-LFA etc.).

In one embodiment, anti-TF antibody of the invention is used for and one or more other treatment antibody joint Use, for example bevacizumabZalutumumab, CetuximabVictibix (Vectibix^TM), ofatumumab, zanolimumab, daratumumab, ranibizumabZenapax, Simulect, Remicade, Humira, Tysabri, Xolair, raptiva, nimotuzumab, Mabthera and/or bent appropriate list It is anti-Other can be used for and the antibody combined treatment antibody for using of the present invention has in the following documents disclosure：WO98/ 40408 (can be with reference to the antibody of natural human TF), WO04/094475 (can be with reference to the antibody of human tissue factor, with normal plasma Control is compared, its blood coagulation that inhibiting factor is not mediated), WO03/093422 is (with TF:The binding affinity of VIIa complexs is more than With the affinity of single TF), or WO03/037361 (for the TF activators or antagonist of Apoptosis associated treatment).

In another embodiment, two or more as the present invention described herein different antibodies be used in combination to treat Disease.Combination of special interest includes two or more non-competing antibody.Therefore, in one embodiment, with here The Antibody Combination in antibody and group II or III as herein defined in defined cross-blocks (cross-block) group I is controlled Treat patient.In another embodiment, suffered from the Antibody Combination treatment in group III with such as the antibody in group II defined below Person.These combination treatments can cause each cell to combine the antibody molecule of greater number, so as to provide higher effect, example The dissolving for such as being mediated by activating complement.

In one embodiment, the administration of anti-TF antibody can be as described below with one or more agent is passed Send and combine, the agent can promote anti-TF antibody or joint composition to reach inside tumor.This method can for example with The delivering of relaxain combines, and wherein (see such as US 6,719,977) relaxain can relax tumour.In an embodiment In, the anti-TF antibody of the present invention can be bonded with cell permeable peptide (CPP).Cell permeable peptide and related peptide be (such as engineering Cell permeability antibody) it is on the books in such as following documents：Zhao et al.,J Immunol Methods.254(1-2), 137-45(2001), Hong et al.,Cancer Res.60(23),6551-6(2000).Lindgren et al., Biochem J. 377(Pt 1),69-76(2004),Buerger et al.,J Cancer Res Clin Oncol.129 (12),669-75(2003),Pooga et al.,FASEB J.12(1), 67-77 (1998) and Tseng et al., Mol Pharmacol. 62(4),864-72(2002)。

In one embodiment, the present invention relates to a kind of disease for treating the cell for being related to express TF in subject The method of disease, the method includes applying the anti-TF antibody and at least one antiinflammatory of therapeutically effective amount to the experimenter of needs.

In one embodiment, this antiinflammatory can be selected from down：Aspirin and other salicylates, Cox-2 (for example brufen, fenoprofen, naproxen, sulindac, double chlorine are fragrant for inhibitor (such as rofecoxib and Sai-Mi-Xi-Bu), NSAID Acid, piroxicam, Ketoprofen, Diflonid, Nabumetone, Etodolac, olsapozine and Indomethacin), anti-IL6R resist Body, anti-IL8 antibody (such as the antibody described in WO2004058797, such as 10F8), anti-IL15 antibody are (for example Antibody described in WO03017935 and WO2004076620), anti-IL15R antibody, anti-CD 4 antibodies (for example Zanolimumab), anti-CD11a antibody (such as efalizumab), anti-α -4/ β -1 integrins (VLA4) antibody (for example that His pearl monoclonal antibody), the CTLA4-Ig for treating inflammation disease, prednisolone, metacortandracin, the antirheumatic of alleviating disease (DMARDs) such as amethopterin, hydroxychloroquine, SASP, pyrimidine synthesis inhibitors (such as leflunomide), IL-1 acceptors Blocking agent (such as anakinra), TNF-α blocking agent (such as Etanercept, infliximab and adalimumab) and class Like medicine.

In one embodiment, such immunosupress and/or immunomodulator can be selected from down：Cyclosporin, Imuran, mycophenolic acid, mycophenolate, corticoid for example metacortandracin, amethopterin, gold salt, SASP, antimalarial, Cloth quinoline that, leflunomide, mizoribine, 15- deoxyspergualins (15-deoxyspergualine), Ismipur, ring phosphorus Acid amides, rapamycin, tacrolimus (FK-506), OKT3, anti-anti-thymocyte globulin, Thymopentin, thymosin extrasin-α and similar Medicine.

In one embodiment, this immunosupress and/or immunomodulator can be selected from immunosuppressive antibody, The antibody for for example being combined with the p75 of IL-2 acceptors, for CD25 antibody (such as described in WO2004045512, such as AB1, AB7, AB11 and AB12), or with such as MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN γ, TNF-α, IL- The antibody that 4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11a or CD58 are combined, or the antibody with their ligand binding.

In one embodiment, this immunosupress and/or immunomodulator can be selected from down：Solvable IL- 15R, IL-10, B7 molecule (B7-1, B7-2, its variant and its fragment), ICOS and OX40, negative T cell regulator inhibitor (such as the antibody of CTLA4) and similar medicine.

In one embodiment, the invention provides a kind of for treating the cell for being related to express TF in subject The method of disease, the method includes applying the anti-TF antibody and anti-C3b (i) antibody of therapeutically effective amount to the experimenter of needs.

In one embodiment, the antibody combined therapeutic agents for using of anti-TF for same treatment disease as described above can To select from down：Histone deacetylase inhibitor (such as PB) and/or DNA renovation agents (for example DNA repair enzymes and It is compositions related, such as dimericine.

For the method including the anti-TF antibody for applying therapeutically effective amount for treating disease as described above of the present invention, Can also include anticancer point to photodynamic therapy (for example anticancer laser therapy-its optionally can using sensitising agent reality Apply, see such as Zhang et al., J Control Release.93(2), 141-50 (2003)), anticancer sound wave and shock wave Therapy is (see such as Kambe et al., Hum Cell.10(1), 87-94 (1997)), and/or anticancer nutritional medication is (see example Such as Roudebush et al., Vet Clin North Am Small Anim Pract.34(1),249-69,viii(2004) And Rafi, Nutrition.20(1),78-82(2004)).Similarly, anti-TF antibody can be used to prepare the light pointed to anticancer Photodynamic therapy (such as anticancer laser therapy-its can optionally use sensitising agent), anticancer sound wave and vibrations wave therapy and/ Or anticancer nutritional therapy is applied to treat the pharmaceutical composition of disease as described above together.

In one embodiment, the invention provides a kind of for treating the disease for being related to TF expression cells in subject The method of disease, the method includes applying the anti-TF antibody of therapeutically effective amount, such as anti-TF of the invention to the experimenter of needs Antibody, and radiotherapy.

In one embodiment, the invention provides a kind of for treatment or the method for pre- anti-cancer, the method includes The anti-TF antibody of therapeutically effective amount, such as anti-TF antibody of the invention, and radiotherapy are applied to the experimenter for needing.

In one embodiment, the invention provides anti-TF antibody, such as anti-TF antibody of the invention is in preparation use In the pharmaceutical composition of the treating cancer being administered in combination with radiotherapy.

Radiotherapy can include radiation or correlation to patient apply radiotherapeutic drug.Radioactive source can be in trouble to be treated (radiotherapy can be for instance in external beam radiation therapy (EBRT) or brachytherapy (BT) for person external or internal Form).Can be used to implementing the radioactive elementary of these methods include such as radium, caesium -137, Iridium-192 source, americium -241, gold - 198th, cobalt -57, copper -67, technetium -99, iodo- 123, iodine -131 and indium -111.

In further embodiment, the invention provides it is a kind of for treatment or pre- anti-cancer method, the method Including the anti-TF antibody combined with surgical operation to the experimenter's administration therapeutically effective amount for needing, such as anti-TF of the invention resists Body.

As described above, the pharmaceutical composition of the present invention can be applied in combination treatment, i.e., with one or more with wait to control Related pharmaceutical agent combinations of the illness for the treatment of, or as other pharmaceutical composition, or by the compounds of this invention with it is a kind of or many Plant additional therapeutic agent co-formulation as above.The compounds of this invention and/or apply medicament altogether that these combination treatments need Dosage can be lower, so as to avoid the possibility toxicity or complication related to various monotherapies.

In addition to upper, other combination treatments interested include as follows：

● for treatment of pancreatic cancer, anti-TF antibody and antimetabolite (such as 5 FU 5 fluorouracil and/or gemcitabine) group Close, may combine from the compound that the following group is selected with one or more：90Y-hPAM4、ARC-100、ARQ-197、AZD-6244、 Bardoxolone methyl, cixutumumab, (IMC-A12), folitixorin calcium, GVAX, easy Puli's nurse agate, KRX-0601, Mei Balong, MGCD-0103, MORAb-009, PX-12, Rh-Apo2L, TLN-4601, trabedersen, Volociximab (M200), WX-671, pemetrexed, rubitecan, Ipsapirone, OCX-0191Vion, 216586-46-8, Lapatinib, matuzumab (matuzumab), Imatinib, Sorafenib (sorafinib), trastuzumab, Exabepilone, Erlotinib (erlotinib), Acrivastine and Cetuximab.

● for colorectal cancer, anti-TF antibody is combined with the compound that one or more are selected from down：Ji Xita Shore, bevacizumab, FOLFOX, FOLFIRI, XELOX, IFL, oxaliplatin, Irinotecan, 5-FU/LV, capecitabine, UFT, EGFR targets agent, such as Cetuximab, Victibix, bundle Shandong wood monoclonal antibody (zalutumumab), Buddhist nun's trastuzumab (nimotuzumab)；VEGF inhibitor or tyrosine kinase inhibitor such as Sutent.

● for breast cancer treatment, anti-TF antibody is combined with the compound that one or more are selected from down：Antimetabolite, Anthracycline antibiotic, taxanes, alkylating agent, Epothilones antihormones (Letrozole (femar), TAM etc.), ErbB2 (Her2/neu) how soft inhibitor (such as Trastuzumab and similar medicine), CAF/FAC be (endoxan (cyclofosfamide), Than star, 5FU) AC (cyclo, doxo), CMF (cyclo, amethopterin, 5FU), Docetaxel+capecitabine, GT (Japanese yews Alcohol, gemcitabine) FEC (cyclo, epi, 5FU) combines with Trastuzumab, the +/- carboplatin of taxol, vinorelbine, Docetaxel, CT is combined with Lapatinib；Capecitabine.

● for bladder cancer treatment, anti-TF antibody is combined with the compound that one or more are selected from down：Antimetabolite (gemcitabine, Alimta (alimta), amethopterin), platinum analogs (cis-platinum, carboplatin), EGFr inhibitor (such as western appropriate former times Monoclonal antibody pricks Shandong wood monoclonal antibody), VEGF inhibitor (such as Acrivastine), Doxorubicin, tyrosine kinase inhibitor it is for example lucky non- For Buddhist nun, trastuzumab, antimitotic agent such as taxanes, such as taxol, and vinca such as vinblastine.

● for prostate cancer therapy, anti-TF antibody is combined with the compound that one or more are selected from down：Hormone/anti- Hormonotherapy；Such as antiandrogen, luteinizing hormone-releasing hormone (LRH) (LHRH) activator, and chemotherapeutics such as taxanes, rice Support anthraquinone, Estramustine, 5FU, vinblastine, Ipsapirone.

● for treatment of ovarian cancer, anti-TF antibody is combined with the compound that one or more are selected from down：Antimitotic Agent, such as taxanes, and vinca alkaloids, pattern Lay (caelyx), Hycamtin.

Diagnostic uses

The anti-TF antibody of the present invention can be also used for diagnostic purpose.Therefore, in further, the present invention relates to Diagnosis composition comprising anti-TF antibody as defined herein.

In one embodiment, anti-TF antibody of the invention can be used for by the level of detection TF or in its film table Level containing the cell of TF on face and the TF expression cells that diagnose activation in vivo or in vitro play actively work in its morbidity Disease.This make testing sample under conditions of complex for example, by being formed between permission antibody and TF, optionally Together with control sample, contact with anti-TF antibody and realize.Then the formation (such as using ELISA) of complex is detected.When with Test sample together using control sample when, two samples are detected with complex, and sample room complex formed it is any The difference of statistically significant indicates the presence of TF in test sample.

Therefore, in further, the present invention relates to it is a kind of for TF antigens in detection sample or expression TF it is thin The method of the presence of born of the same parents, including：

- formed under conditions of complex between permission antibody and TF, make the anti-TF antibody or the present invention of sample and the present invention Bispecific molecule contact；With

- analyse whether to define complex.

In one embodiment, the method is implemented in vitro.

More specifically, the invention provides for identifying, diagnose aggressive cell and tissue, and other are by of the invention The method of the cell that anti-TF antibody is targeted, and for state, the danger of generation cancer after the progress, the treatment that monitor therapeutic treatment The method of danger, cancer progression etc..

In an example of this diagnostic assay method, the invention provides diagnosing the side of aggressive cellular level in tissue Method, is included between anti-TF antibody and the potential tissue containing TF and forms immunocomplex, and the formation of detection immunocomplex, The wherein formation of immunocomplex is related to the presence of invasion cell in tissue.Contact can use labeled separation antibody and Standard imaging techniques are implemented in vivo, or can implement on tissue sample in vitro.

Anti- TF antibody can be used for by the peptide containing TF and peptide in any suitable biological sample of any suitable technology for detection Fragment.The example of the routine immunization determination method that the present invention is provided includes, but not limited to ELISA, RIA, FACS measure, plasma Resonance measure, chromatographic determination, histogenic immunity group, western traces, and/or the immunoprecipitation using anti-TF antibody.This Bright anti-TF antibody can be used to detect TF the and TF fragments from people.For the anti-TF antibody that uses in these techniques and/ Or the appropriate flags thing of SA includes, but not limited to various enzymes, prothetic group, fluorescent material, luminescent substance and radioactivity thing Matter.The example of suitable enzyme includes horseradish peroxidase, alkaline phosphatase, beta galactosidase or acetylcholinesterase；Close The example of suitable prosthetic group complexes includes streptavidin/biotin and avidin/biotin；The example of suitable fluorescent materials includes umbrella Shape ketone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine base amine fluorescein (dichlorotriazinylamine Fluorescein)/dansyl Cl or phycoerythrin；The example of luminescent substance includes luminol；And suitable radioactive materials Example include¹²⁵I,¹³¹I,³⁵S, and³H。

Anti- TF antibody can be marked with detectable substrate to utilize by competition method of immunity in biological sample TF peptides standard and unlabelled anti-TF antibody are measured.In this measure, by biological sample, labeled TF peptide standards Mix with anti-TF antibody, and detect the amount of the labeled TF standard peptides combined with unlabelled anti-TF antibody.Biological sample The amount of middle TF antibody with and the amount of labeled TF standards that combined of anti-TF antibody be inversely proportional to.

Anti- TF antibody is particularly useful in terms of the in-vivo imaging of tumour.The tumour in-vivo imaging related to TF can pass through Any suitable technology is implemented.For example, can use⁹⁹Tc- labelling methods radiate gamma-ray isotopic mark using another kind of The anti-TF antibody of notation, the anti-TF antibody come in marked tumor or the second mark (such as FITC marks) from tumour: TF complexs, and with γ scintillation camera (such as Elscint Apex 409ECT devices) be imaged, typically using low energy, High-resolution collimator or the all-round collimator of low energy.It is then possible to the tissue to being colored carries out radioactive counts assessment, As the instruction of the amount of intra-tumor TF- related peptide.The image obtained by using these technologies can be used for assessment patient, lactation and move Thing or tissue in TF bio distribution (for example when using TF or TF fragments as the presence of invasive carcinoma cell biological marker When).The version of this technology includes providing the imaging better than gammacamera technology using magnetic resonance imaging (MRI).Class As immunoscintigraphy method and principle it is on the books in such as following documents：Srivastava (editor), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum publishing houses 1988), Chase,"Medical Applications of Radioisotopes,"in Remington's Pharmaceutical Sciences, the 18th edition, Gennaro et al., (editor), the 624-652 page (Mack Publishing Co., 1990), And Brown, " Clinical Use of Monoclonal Antibodies, " in Biotechnology And Pharmacy 227-49, Pezzuto et al., (editor) (Chapman＆Hall 1993).These images can be also used for other anticancers The targeted delivery of agent, the example of other anticancers has been described here (such as apoptosis agent, toxin or the combination of CHOP chemotherapy Thing).And/or these images are also used as the basis of the surgery operating technology for removing tumour.And, this in-vivo imaging Technology can allow it is identified with tumour (due to the presence of other biological markers, transfer etc.) in patient, but tumour cannot Identification and positioning tumor in the case of being identified with conventional analytical technology.All these methods are all the features of the present invention.

The in-vivo imaging and other diagnostic methods that the present invention is provided be particularly useful in detecting people patient (for example previously not by Patient of the diagnosis with cancer or in cancer recovery/paracmastic patient) internal micrometastasis.For example, whole cancers are accounted for thin Born of the same parents are up to 90% carcinoma (Carcinoma) cancer cell and have been proved to can use anti-TF antibody couplings composition well to dye.With The detection of monoclonal described here anti-TF antibody can indicate the presence of aggressiveness/invasion epithelial tumor, or/and carry Instruction for using the feasibility of the anti-TF antibody of irrelevant monoclonal for these micrometastasis.

In one embodiment, the invention provides in-vivo imaging method, wherein by the anti-TF antibody of the present invention and inspection Survey-promote radiopaque reagent (detection-promoting radio-opaque agent) to be coupled, coupled antibody is applied With to host's (such as in by being expelled to blood flow), and determine the presence of labelled antibody and positioning in host.By this technology With any other diagnostic method provided herein, the invention provides for screening in people patient body or from people patient obtain life The method of the presence of disease associated cell in thing sample.

For diagnosing image, radio isotope can direct or through using intermediate function group indirectly with it is anti- TF antibody connects.Useful intermediate function group includes chelating agent, and such as ethylenediamine tetra-acetic acid and diethylenetriamine pentaacetic acid are (see example Such as US 5,057,313).

In addition to radio isotope and radiopaque reagent, diagnostic method can with dyestuff (for example with biotin- Streptavidin complex is coupled), contrast preparation, fluorescent chemicals or molecule and for magnetic resonance imaging (MRI) reinforcing agent (for example Paramagnetic ion) (see such as US Pat.No.6,331,175, which describe MRI technique and the antibody that is coupled with MRI reinforcing agents Prepare) be coupled anti-TF antibody implement.This diagnosis/detection reagent can be from for the reagent of magnetic resonance imaging and Fluoresceinated Select in compound.In order that anti-TF antibody load radioactive metal or paramagnetic ion, it may be necessary to make it and there is long afterbody Reagent reacting, multiple chelation groups for ions binding are overlapped with the afterbody.This afterbody can be polymer, example As poly-D-lysine, polysaccharide or other have can be combined with chelation group suspend in midair group derivatizations or can derivatization chain, Described chelation group such as porphyrin, polyamines, crown ether, double amithiozones (bisthiosemicarbazone), poly- oxime, and it is known Can be used for the similar group of this purpose.Chelating agent can be with standard chemical method and anti-TF antibody couplings.

Therefore, the invention provides diagnostic anti-TF antibody coupling matters, wherein anti-TF antibody and contrast preparation (for example for Magnetic resonance imaging, computed tomography or ultrasound contrast reinforcing agent) or radionuclide conjugation, radionuclide can be example Such as launch the isotope of γ, β, α, auger electrons or positive electron.

In further, the present invention relates to be used for the presence of the cell of TF antigens or expression TF in detection sample Kit, including

The anti-TF antibody of-present invention or the bispecific molecule of the present invention；With

The operation instructions of-kit.

In one embodiment, the invention provides the kit for diagnosing cancer, including containing anti-TF antibody Container, and be used to for one or more detect the reagent that anti-TF antibody is combined with TF peptides.Reagent can include such as fluorescence labels, Enzyme label or other detectable labels.Reagent can also include second or the 3rd antibody or reagent for enzyme reaction, wherein enzyme Reaction generation can be by visual product.In one embodiment, the invention provides a kind of diagnostic kit, it includes putting The anti-TF antibody of one or more present invention in labeled or unlabelled form in suitable container, for It is used for the reagent and substrate or derivatization reagent for being detected in this measure of incubation in indirect determination, these examinations Agent depends on the property of label.Contrast agents and operation instructions can also be included.

Diagnostic kit can also be provided to utilize anti-TF antibody, the anti-TF antibody for being for example coupled/marking, detection tissue The presence of sample or host's inner cell activity or detection TF peptides.In this diagnostic kit, and herein it locate the use of record In the kit for the treatment of use, anti-TF antibody is typically placed in container with lyophilized form, be provided separately or with it is extra The special antibody for target cell or peptide is provided together.Typically, also including pharmaceutically acceptable carrier (such as inert diluents Agent) and/or its component such as Tris, phosphate or carbonate buffer agent, stabilizer, preservative, biocide, biocide, Inert protein such as seralbumin, (being typically positioned in other container for mixing), and extra reagent is (typically It is also placed in other container).In some kits, also including the SA that can be combined with anti-TF antibody, it leads to Often it is placed in other container.SA is generally coupled with label, and in the way of similar to anti-TF antibody of the invention Prepare.Using method above-mentioned and that herein it locates record, it is possible to use anti-TF antibody limiting the subclass of cancer/tumour cell, With these cells of sign and linked groups/growth-gen.

By taking out tissue samples, and the combination of the anti-TF antibody to this sample offer mark of the invention from patient, In situ detection can be realized.The offer of the anti-TF antibody of the present invention by applying (apply) to biological sample or can be covered (overlay) the anti-TF antibody of mark of the invention is realizing.By using this program, not only it is possible to establish that TF or TF pieces The presence of section, it is also possible to determine these peptides in the distribution (such as when the diffusion of cancer cell is assessed) in detecting tissue.Utilize The present invention, those of ordinary skill can easily appreciate that, (can for example dye journey to any one of a large amount of Histological methods Sequence) changed to realize this in situ detection.

In further, the present invention relates to a kind of anti-idiotype, it is anti-with the present invention as described herein TF antibody is combined.

Anti- idiotype (Id) antibody is following antibody, and its identification is typically only with what the antigen binding site of antibody was associated Special determinant.Id antibody can be by being prepared, wherein the thing of the animal with the anti-TF monoclonal antibodies immune animal that prepare anti-Id Plant and hereditary form is identical with species and hereditary form that the anti-TF monoclonal antibodies are originated.Use usually can be recognized by the animal of immunity Carry out the idiotypic determinant of immune antibody, and by produce the antibody (anti-Id antibody) for these idiotypic determinants come It is responded.This antibody is on the books in such as US 4,699,880.This antibody is further characteristic of the invention.

Anti- Id antibody also acts as " immunogene " and immune response is induced in another animal, produces so-called anti- Id antibody.Anti- Id antibody may be identical in epi-position with the initial monoclonal antibody for inducing anti-Id antibody.Therefore, by using pin Antibody to the idiotype determinant of certain monoclonal antibody, it is possible to identify other expression with mutually homospecific antibody gram It is grand.Anti- Id antibody can be changed (produce anti-Id antibody variants whereby) and/or derivatization by any suitable technology, For example herein at it with regard to the technology described by anti-TF antibody of the invention.For example, anti-Id monoclonal antibodies can be with carrier such as keyhole Hemocyanin (KLH) is coupled, and for immune BALB/c mouse.Anti- Id is usually contained from the serum of these mouse to resist Body, it has the binding property of (even if differing) similar with original/parent's TF antibody.

The present invention is further illustrated by examples below, but they are not construed as further restriction.

Embodiment

Embodiment 1

The expression construct of tissue factor (TF)

Generate for expressing TF or the complete codon optimised constructs of its extracellular domain in HEK, NS0 or Chinese hamster ovary celI. The protein encoded by these constructs is identical with Genbank accession number NP_001984 of TF.The construct contains suitable use In clone restriction site and optimum Kozak sequences (Kozak, 1987).The construct is cloned in into mammal expression to carry In body pEE13.4 (Lonza Biologics) (Bebbington, Renner et al.1992), pEE13.4TF is obtained.Use PCR is from the part of synthesis construct amplification coding TF extracellular domains (ECD) (amino acid/11-251), while addition one contains 6 The C-terminal His label (TFECDHis) of histidine residues.The construct is cloned in pEE13.4, and is sequenced completely with true Recognize the correctness of construct.

Embodiment 2

The transient expression in HEK-293F cells

Freestyle^TM(HEK-293 of a kind of adaptation suspension growth and chemically defined Freestyle culture mediums is sub- for 293-F Clone, (HEK-293F)) cell obtains from Invitrogen, and with 293fectin (Invitrogen) making according to manufacturer Transfected with suitable DNA with explanation.In the case of antibody expression, coexpression as described in example 10 above suitable Heavy chain and light chain vector.

Embodiment 3

The semi-stability expression in NS0 cells

PEE13.4TF is stably transfected in NS0 cells, and without 7.5 μM of methyl sulfoxide Asias of glutamine and presence Growth under conditions of amine (MSX) selects stable clone.Clone pool (pool of clones) is allowed to grow in suspension medium, Selection pressure is kept simultaneously.The TF expression in pond is tested by facs analysis, and saves standby from damage.

Embodiment 4

Stably express in Chinese hamster ovary celI

PEE13.4TF is stably transfected in CHO-K1SV (Lonza Biologics) cell, and without glutamine Stable clone is selected with there is growth under conditions of 50 μM of MSX.Picking monoclonal, expand and by FACS as described below Analysis carries out TF expression inspections.Select high-expression clone and save standby from damage.

Embodiment 5

Purifying with His label TF

TFECDhis is expressed in I HEK-293F cells.His labels in TFECDHis are caused with fixed metal parent Purifying is carried out with chromatogram to be possibly realized.In this process, Co on the chelating agent band that is fixed on chromatography resin is made²⁺Cation.Will In batch mode (ie in solution) is incubated supernatant containing TFECDHis with resin.Protein with His- labels and resin beads Strength is combined, and being present in the other oroteins in culture supernatant then will not strongly combine.After incubation, return from supernatant Pearl is received, and is filled in pillar.Clean pillar to remove the protein of weak binding.Then with the buffer solution elution containing imidazoles The strong TFECDHis albumen for combining, wherein imidazoles and His competition binding Co²⁺.By the enterprising row buffering liquid of desalting column exchange from Eluent is removed in protein.

Embodiment 6

Transgenic mice immune programme for children

People's monoclonal antibody mouse was per 14 days alternating 5x10⁶NS0-TF cells of semi-stability transfection or with 20 μ g TFECDHis eggs White matter carries out immunity.Implement 8 immunity, 4 times intraperitoneal (IP) immunity and 4 root of the tail subcutaneous (SC) immunity altogether.Using cell First time immunity in complete Freund's adjuvant (CFA；Difco Laboratories, Detroit, MI, USA) in complete.For All other immunity, cell IP injections in PBS, and TFECDHis cannots be used up full Freund's adjuvant (IFA；Difco Laboratories, Detroit, MI, USA) SC injections.It is (special in antigen as described in example 7 above when serum titer is abundant Find the serum of 1/50 or lower dilution in sun at least 2 times continuous double week filter events in different in nature screening test method Property), carried out within 4 and 3 days before fusion with 10 μ g TFECDHis albumen being dissolved in 100 μ l PBS extra intravenous twice (IV) booster immunization (boosted).

Carried out in CFA using the first time immunity of cell, immune for all other each (7), cell is in PBS IP is injected.When finding that serum titer is abundant, 4 and 3 days before fusion, the mouse 1x10 being dissolved in 100 μ l PBS⁶It is individual instantaneous The extra booster immunizations of semi-stability transfection NS0-TF cell IV 2 times.

Embodiment 7

Isogeneic specificity screening is determined

Anti- TF in the serum or people's monoclonal antibody (human monoclonal antibodies) hybridoma or transfectoma cultures supernatant of Jing immune mouses The presence of antibody determines (four fractions) and uses Fluorometric Micro volume by isogeneic specificity screening Assay Technology(FMAT；Applied Biosystems, Foster City, CA, USA) determined.

For this purpose, being applied in combination 3 measurement systems and 1 measurement system based on pearl based on cell.Based on cell Measure in, it is determined that with TH1015-TF (the HEK-293F cells of transient expression TF；As described above produce) and A431 (its TF is expressed on cell surface) and HEK293 wild-type cells (not expressing TF, negative control) combination.In the survey based on pearl In fixed, the combination to being coupled to the biotinylated TF (SB1015-TF) of streptavidin pearl is determined.

Sample is added to cell/pearl to carry out TF combinations.Subsequently, with fluorescence conjugate (goat anti-human IgG-Cy5； Jackson ImmunoResearch) detection and people's monoclonal antibody combination.Mouse Anti-Human TF antibody (ERL；Genmab companies with Alexa-647 is coupled) it is used as positive control, people's list anti-mouse combining anteserum (pooled serum) and mouse-chrompure- Alexa647 antibody is used as negative control.Sample is entered with the cell detecting systems of Applied Biosystems 8200 (8200CDS) Row scanning, and with " counting x fluorescence " as reading.

Embodiment 8

People's monoclonal antibody hybridoma is generated

People monoclonal antibody mouse to there is (as defined above) with abundant antigentic specificity titre sentences euthanasia, collects spleen With abdominal aorta and the lymph node of vena cave both sides.By electro' asion electro' asion system (the Cyto Pulse of CEEF 50 Sciences, Glen Burnie, MD, USA) carry out splenocyte and LNC basically according to the operation instruction of manufacturer With the fusion of mouse myeloma cell line.The selection and culture of people's monoclonal antibody hybridoma of gained is carried out (for example according to standard method It is described in the following documents：Coligan J.E.,Bierer,B.E.,Margulies,D.H.,Shevach,E.M.and Strober,W.,eds.Current Protocols in Immunology,John Wiley&Sons,Inc.,2006)。

Embodiment 9

The mass spectrum of the antibody of purifying

The supernatant (every part of 0.8ml) that aliquot from 6 holes or Hyperflask platforms is contained into antibody is with containing Protein G The PhyTip posts (PhyNexus Inc., San Jose, USA) of resin are in the work station (Caliper of Sciclone ALH 3000 Lifesciences, Hopkinton, USA) on purified.The use of PhyTtip posts according to the operation instructions of manufacturer, But buffer solution combination buffer PBS (B.Braun, Medical B.V., Oss, Netherlands) and elution buffer (the Fluka Riedel-de of 0.1M glycine-HCl pH 2.7Buchs, Germany) replace.After purification, sample is used 2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands) are neutralized.Or, in certain situation Under, large volume of culture supernatant is purified with albumin A affinity column chromatography.

After purification, sample is placed in 384- orifice plates (Waters, 100ul square hole plate, part number 186002631).Sample With N- glycosylase F, (Roche lot numbers 11365177001 overnight remove glycosyl at 37 DEG C.Addition DTT (15mg/ml) (1 μ l/ holes) and 1h is incubated at 37 DEG C.Sample (5 or 6ul) is in Acquity UPLC^TMBEH300C18,1.7 is used on (Waters, Milford, USA) μm, 2.1x50mm posts carry out desalination at 60 DEG C.Using MQ water and LC-MS level acetonitriles (Biosolve, lot number 01204101, Valkenswaard, The Netherlands), both of which containing 0.1% formic acid (Fluka, lot number 56302, Buchs, Germany) respectively as eluent A and B.In the micrOTOF operated with cation mode^TMMass spectrograph (Bruker, Bremen, Germany online record flight time LC-MS spectrometry on).Before analysis, pre-mixing agent (ES is adjusted with ES Tuning mix) (Agilent Technologies, Santa Clara, USA) calibration 900-3000m/z scales.Mass spectrum is used DataAnalysis^TMV.3.4 (Bruker's software) is deconvoluted, using maximum entropy algorithm (Maximal Entropy Algorithm the molecular weight between 5-80kDa) is found.

After deconvoluting, the heavy chain and light chain quality to the whole samples of gained is compared, with the antibody for finding to repeat. In the comparison of heavy chain, there may be for C- ends lysine variant is accounted for.As a result a series of antibody of uniquenesses has been obtained, The definition of wherein " uniqueness " is the unique combination of heavy chain and light chain.In the case where finding to repeat antibody, using from other surveys The result of examination determines which is to continue with the optimal material tested.

MS analyses are carried out to the heavy chain and light chain molecule amount of 118 TF specific hybridization knurls, the antibody of 70 uniquenesses is obtained (unique heavy chain/light chain combination).They are characterized in some functional tests, are identified 14 leading candidates, TF specific antibodies.

Embodiment 10

The sequence analysis of anti-TF people's monoclonal antibody variable domain and to the clone in expression vector

From 5x10⁶Individual hybridoma prepares the total serum IgE of anti-TF people's monoclonal antibody, and uses SMART from 100ng total serum IgEs RACE cDNA amplification kits (Clontech) prepares 5 '-RACE- complementary DNAs (cDNA) according to the operation instructions of manufacturer. VH (weight chain variable district) and VL (light chain variable district) code area is expanded by PCR, and with Zero Blunt PCR Cloning Kits (Invitrogen) in being cloned into pCR-Blunt II-TOPO carriers (Invitrogen).For everyone monoclonal antibody, to 16 VL Clone and 8 VH clones are sequenced.Sequence is given in the sequence table of this paper and Fig. 1.

Table 1A and table 1B (below) give the overview of antibody sequence information and most homologous Germline sequences.

Table 1A heavy chain homologues

Table 1B light chain homologues

Sequence table is referred to:

Embodiment 11

Antibody purification

Culture supernatant is filtered by 0.2 μm of cecum filter (dead-end filter), and is loaded into 5ml albumin As On post (rProtein A FF, Amersham Bioscience), with 0.1M citric acid-NaOH, pH 3 is eluted.Eluate is immediately 2M Tris-HCl, pH 9 is used to neutralize, and dialyzed overnight is to 12.6mM NaH₂PO₄,140mM NaCl,pH 7.4(B.Braun) In.After dialysis, sample is filtered by 0.2 μm of cecum Filter Sterile.Purity is determined by SDS-PAGE, and by turbidimetry and 280nm absorbance measuring concentration.The antibody of purifying is divided into into decile, and is stored in -80 DEG C.Once melt, by the antibody of purifying Decile is stored in 4 DEG C.Implement mass spectrum to identify the molecule of the heavy chain of antibody and light chain expressed by hybridoma as described in Example 9 Amount.

Embodiment 12

Antibody cross competition research is carried out with sandwich-ELISA

Overnight it is coated with+4 DEG C with the every kind of anti-TF people's monoclonal antibody (the μ L/ holes of 0.5 or 2 μ g/ml 100) being diluted in PBS ELISA plate wells.With PBS ELISA holes, be dissolved in PBS 2% (v/v) chicken serum (Gibco, Paisley, Scotland) close 1 hour at room temperature, and cleaned again with PBS.Subsequently, the anti-TF people's monoclonal antibodies (10 μ g/mL) of 50 μ L are added, Then add 50 μ L TFECDHis (0.5 or 1 μ g/ml) (to produce in Genmab；Embodiment 5), and RT incubate 1 hour (while Concussion).Flat board is cleaned 3 times with PBST (PBS+0.05% tweens), and uses 1:The anti-his biotins BAM050 of 2000 dilutions exists RT is incubated 1 hour (while concussion).Cleaning flat board, and with streptavidin-poly- HRP (Sanquin, Amsterdam, The Netherlands) incubate 20 minutes in RT, clean again.It is right with ABTS (Roche Diagnostics) under RT in the dark Reactant further develops the color, and by addition 2% (w/v) Oxalic acid stopps reaction after 15 minutes, and measures 405nm absorbances.

Table 2 shows, it is possible to authenticate (contend with one other combination to go out 3 cross-blocks groups (cross-block group) The antibodyome of TFECDHis), wherein antibody 013,044 and 087-Lg63 belongs to a cross-blocks group (group I), antibody 011, 017-D12,42,092-A09 and 101 belongs to another cross-blocks group (group II), and antibody 003,025,109 and 111 belongs to the Three cross-blocks groups (group III).Antibody 114 is found to be combined with the antibody competition from cross-blocks group II and III TFECDHis.The combination of antibody 098 and TFECDHis can be by the antibody competition from cross-blocks group II and III.

Table 2-combination of the anti-TF antibody competitions to TFECDHis.

White edge is represented and does not compete the combination to TFECDHis, and light grey frame represents knot of the partial competition to TFECDHis Close, Dark grey frame represents combination of the competition to TFECDHis.

Embodiment 13

The combination of anti-TF people's monoclonal antibody and TF extracellular domains in ELISA

The specificity of the anti-TF people's monoclonal antibody of gained is assessed with ELISA.Elisa plate (Microlon；Greiner Bio-One) With PBS is dissolved in, the 0.5 μ g/mL TFECDHis of pH 7. are overnight coated with+4 DEG C.Empty coated ELISA flat boards, and with molten 2% (v/v) chicken serum (Gibco, Paisley, Scotland) room temperature in PBS is closed 1 hour, and with telling containing 0.05% PBS (PBST) cleanings of temperature 20.Subsequently, (2% (v/v) chicken serum and 0.05% (v/v) Tween-20 will be supplemented with PBSTC PBS RT is incubated 1 hour under people's monoclonal antibody earthquake state (300rpm) being serially diluted in).With 1:5,000 are diluted in PBSTC HRP- be coupled goat anti-human's IgG antibody (Jackson ImmunoResearch) earthquake state (300rpm) under RT temperature Educate 1 hour, to detect people's monoclonal antibody of combination.In the dark one is entered to reactant with ABTS (Roche Diagnostics) under RT Step colour developing, and then 405nm absorbances are measured by addition 2% (w/v) Oxalic acid stopps reaction after 15-30 minutes.Employment list Anti- KLH (for the human monoclonal antibodies of KLH (keyhole limpet hemocyanin)) is used as negative control.Mouse Anti-Human TF (ERL) is used as Positive control (anti-mouse IgG of HRP marks is used as conjugate).Using GraphPad Prism V4.03 softwares with non-linear Return (the S types dosage-response of variable slope) analysis binding curve.

As seen in Figure 3, all anti-TF combine TFECDHis.EC to people's monoclonal antibody₅₀Value is the mean value of 3 experiments, is 0.09-0.46nM does not wait (table 3 sees below).

Table 3:

Embodiment 14

The combination of anti-TF people's monoclonal antibody and film combination TF

The combination of anti-TF people's monoclonal antibody and film combination TF is measured by facs analysis, using TF transfect Chinese hamster ovary celI or The tumor cell line MDA-MB-231, (fluorescein transfection) A431 and Bx-PC3 of expression TF.

Cell is suspended in into (2x10 in PBS⁶Cell/ml), in being placed in 96 hole V- base plates (50 μ l/ holes).Add to cell Plus 50 μ l be serially diluted in FACS buffer solution (addition 0.1%BSA and 0.02% sodium azide PBS) in people's monoclonal antibody, and Incubated on ice 30 minutes.After cleaning 3 times with FACS buffer solution, add the goat anti-human IgGFc that 50 μ l phycoerythrin (PE) are coupled (Jackson ImmunoResearch) is (with 1:100 are diluted in FACS buffer solution).It is (dark after standing for 30 min on ice In), cleaning cell 3 times, and by the spy of Flow cytometry people's monoclonal antibody on FACSCalibur (BD Biosciences) Different combination.People's monoclonal antibody-KLH is used as negative control.With the anti-TF of mouse and anti-mouse IgGFc of subsequent PE- couplings as the positive Control.Using GraphPad Prism V4.03 softwares (GraphPad Software, San Diego, CA, USA) with non-linear Return (the S types dosage-response of variable slope) analysis binding curve.

Fig. 4 shows the example of the special people's monoclonal antibody of TF- and the binding curve of MDA-MB-231 cells.Table 4 gives The Chinese hamster ovary celI (S1015-TF) that the special people's monoclonal antibodies of TF- are transfected with TF, the combination of MDA-MB-231, A431 and Bx-PC3 cell The general view of EC50 values.

Table 4-by the EC50 and maximum of the combination of the TF- specific humans monoclonal antibody and different cell types of facs analysis determination The general view of Mean Fluorescent Index (maximum MFI) value.

EC50 values are in terms of nM.To MDA-MB-231, the maximum MFI of BxPC3 and A431 cells is 30 μ g/mL antibody, for S1015-TF is 7.5 μ g/mL antibody.

Embodiment 15

FVIIa is suppressed to be combined with TF

The suppression that TF- people's monoclonal antibody is combined to FVIIa with TFECDHis is measured by ELISA.With TFECDHis (0.5 μ g/ ML, 100 Μ l/ holes) overnight it is coated with ELISA flat boards.Turned letter flat board, the use closings of the PBS containing 2% (v/v) chicken serum (1 hour, RT), turn again.Add the 4- times of TF- people's monoclonal antibody being serially diluted or people monoclonal antibody-KLH (negative control) to hole, subsequently addition The FVIIa of EC50 concentration (100nM), flat board is incubated 1 hour (while concussion, 300rpm) in RT.Cleaning flat board, and with as above Rabbit-anti-FVIIa (2.5 μ g/mL；Abcam) incubate.Cleaning flat board, and with pig-anti-rabbit igg-HRP antibody (1:2,500； DAKO) incubate.After cleaning, immunocomplex is manifested as substrate with ABTS.By adding the reaction of 2%v/v Oxalic acid stopps, subsequently The optical density of 405nm is measured with ELISA readers.Calculated with GraphPad prism (nonlinear regression analysis) and obtain 50% suppression AC needed for system (IC50).

Fig. 5 shows that the antibody from cross-blocks group II and III efficiently inhibits the combination of FVIIa and TF, The antibody of Self-crossover blocking I groups can not suppress FVIIa to combine (or inhibition level is much lower).

Table 5 shows the IC50 and maximum suppression value (percentage) of the suppression that the special people's monoclonal antibodies of FT are combined to FVIIa with TF.

The IC50 values and maximum suppression value (percentage) of the suppression that-TF- specific humans monoclonal antibody of table 5 is combined to FVIIa with TF

Embodiment 16

The suppression of the ERK phosphorylations of FVIIa inductions

When proconvertin a (FVIIa) is combined with TF, trigger mitogen-activated kinases (p42 and p44MAPK or ERK1 and ERK2) phosphorylation.Squamous epithelium cancerous cell line A431 expresses high-caliber TF, and according to AlphaScreen Surefire ERK measurement systems (Perkin Elmer) are measured, after being stimulated with FVIIa, induction of optimal in 10 minutes The ERK phosphorylations (ERK-P) of (3-5 times).

A431 cells (30,000 cells/wells) are inoculated in 96 hole TC plates, and (contain 20% in serum free medium The RPMI of HAS and penicillin/streptomycin) in overnight incubation (37 DEG C, 5%CO₂, 85% humidity).Then it is (no added with DMEM Agent) replace culture medium, and by cell culture 1.5 hours.3 times of TF- people's monoclonal antibodies being serially diluted of addition or people monoclonal antibody-KLH, and will Cell culture 0.5 hour.Then, cell (50nM is stimulated with EC80 concentration with FVIIa；10 minutes；37 DEG C, 5%CO₂, 85% is wet Degree).With cell of PBS, and cracked with 25 μ L lysis buffers (Perkin Elmer, Surefire kit).It is right Lysate is centrifuged (3 minutes, 330x g, RT).4 μ L of supernatant are transferred to into (Perkin in 384 hole Proxiplate Elmer).Add reaction buffer/work that 7 μ L contain AlphaScreen pearls (Perkin Elmer, Surefire kit) Change buffer solution pre-composition, in RT Incubate plates 2 hours under dark.With " the Surefire of EnVision technology Plus " codes read flat board.

Fig. 6 shows that measured with AlphaScreen Surefire ERK measurement systems, antibody 013 does not suppress FVIIa to induce ERK phosphorylations, 044 and 111 medium suppression ERK phosphorylations, and all other antibody can efficiently block ERK phosphorylations.

Table 6 shows the IC50 values and maximum suppression value (percentage of the ERK phosphorylations that the special people's monoclonal antibodies of TF- are induced FVIIa Than), measured using AlphaScreen Surefire ERK measurement systems.

The ERK phosphorylations that table 6-TF- specific humans monoclonal antibody is induced FVIIa (use AlphaScreen Surefire ERK Measurement system measure) IC50 values and maximum suppression value (percentage)

The result of gained is in addition true by Western blot analysis in AlphaScreen Surefire ERK are determined Recognize, using HaCaT and BxPC3 clones.30,000 cells/wells are seeded in into (the starvation trainings of the DMEM containing Cmin serum Foster base) in, and incubated overnight.Cell is further cultivated 2 hours in the DMEM of serum-free, is added in last 30 minutes of culture Plus anti-TF antibody.0,10 or 50nM FVIIa 10 minutes (37 DEG C) of stimulation of cell, subsequently cracking is (every in cell pyrolysis liquid The μ L lysis buffers of hole 50, crack 30-60 minutes, RT under the conditions of earthquake).To each sample sample of the addition containing 25 μ L SDS Savor buffer solution.Sample is loaded on PAGE gel, is run, and be blotted with standard western blot program.Trace Closed 1 hour under RT with the TBST 1x containing 5% unrelated protein (ELK).With rabbit-anti-ERK-P antibody incubation traces (O/N, 4 ℃).Trace is cleaned with TBST 1x, and (1 hour, RT) is incubated with anti-rabbit igg HRP, HRP Substrate developments are used in cleaning, are used Optigo Ultima imaging systems (Isogen Life Sciences) are imaged.

Fig. 6 shows the result of a subgroup (sub-panel) of antibody in BxPC3 cells.10nM FVIIa inductions ERK phosphorylations are not suppressed by antibody 013, but (the latter is as described herein by the effectively suppression of antibody 111,044 and 025 One example of every other TF- specific humans monoclonal antibody).The ERK phosphorylations (50nM FVIIa) of more induced strong are not by antibody 013rd, 111 and 044 suppresses, but is suppressed by antibody 025.

Embodiment 17

Suppression to the IL-8 releases of FVIIa inductions

Suppress the ability of the IL-8 releases of FVIIa inductions with MDA-MB-231 cell tests TF specific humans monoclonal antibody.Will be thin Born of the same parents are inoculated in 96 orifice plates (60,000 cells/well), and containing CS, Sodium Pyruvate, l- glutamine, MEM NEAA and mould Cultivate in the DMEM of element/streptomysin (O/N, 37 DEG C, 5%CO₂).Tissue culture medium (TCM) is removed, (is contained in serum-free, high-calcium medium Have the DMEM of penicillin/streptomycin) in cleaning 2 times, 105 minutes are further cultured in this culture medium.It is anti-that addition is serially diluted Body and by cell culture 15 minutes.Addition FVIIa (Novo Nordisk；Final concentration 10nM), by cell culture 5 hours.Remove Supernatant is simultaneously centrifuged (300x g, RT).Supernatant is measured according to the scheme (Sanquin) of manufacturer with IL-8ELISA kits The concentration of middle IL-8.

Fig. 7 shows, from the antibody of cross-blocks group II and III the MDA-MB-231 cells that FVIIa induces efficiently are suppressed IL-8 releases, but except the antibody 111 of meridian genomics III.From antibody (013,044 He of cross-blocks group I 87-Lg6) can not all suppress the IL-8 releases that FVIIa is induced.

Table 7 shows that TF- specific humans monoclonal antibody suppresses the IC50 values and maximum suppression value of the IL-8 releases of FVIIa inductions (percentage).

Table 7-TF- specific humans monoclonal antibody suppresses the IC50 values and maximum suppression value (percentage of the IL-8 releases of FVIIa inductions Than).

Embodiment 18

FXa is suppressed to generate

The ability that TF specific humans monoclonal antibody suppresses FXa to generate is tested in following measurement system, wherein special with colorimetric FXa Lower transformations of the FX to FXa of different in nature substrate measurement TF/FVIIa complexs effect.To flat 96 orifice plate addition TF (Innovin) and TF specific human monoclonal antibodies, positive control (the anti-TF of mouse) and the negative control (people monoclonal antibody-KLH) being serially diluted (all dilutes Containing 3mM CaCl₂Hepes buffer solutions in).Flat board is incubated 30 minutes under RT, and add FVIIa (final concentration 1nM) and FX(ERL；Final concentration 200nM).Flat board is incubated 30 minutes at 37 DEG C.From every hole take 50 μ l be transferred to it is (molten containing stop buffer 5mM EDTA in 100ml Hepes buffer solutions) (preheating, 37 DEG C) 96 orifice plates in.Addition FXa specific substrates Chromogenix-2765 (Instrumation Laboratory Company), flat board is incubated 60 minutes at 37 DEG C, and is surveyed The OD405nm of 37 DEG C of amount.

Fig. 8 shows that antibody 017-D12 strong inhibitions FXa are generated, and 013 shows medium suppression, and other antibody are generated to FXa Show relatively low suppression or no suppression.

Table 8 shows that TF- specific humans monoclonal antibody suppresses the IC50 values and maximum suppression value (percentage) of FXa generations.

Packet	People monoclonal antibody TF	IC50nM	% maximum suppressions
				I	13	0.05	31
I	44	NA	3
				I	87-Lg6	nt	nt
II	11	0.05	26
				II	017-D12	0.28	84
II	42	nt	nt
				II	092-A09	0.30	21
II	101	nt	nt
				II/III	98	0.43	14
II/III	114	0.24	21
				III	3	0.07	21
III	25	0.30	19
				III	109	0.09	18
III	111	0.07	7

The special people's monoclonal antibodies of table 8-TF- suppress the IC50 values and maximum suppression value (percentage) of FXa generations.

Embodiment 19

Suppress blood coagulation

With suppression of the measurement system measurement TF- people's monoclonal antibody in the clotting time for determining TF inductions to blood coagulation.In 96 orifice plates Prepare following mixture：17μl 100mM CaCl₂(final concentration 17mM), 10 μ l 1:100innovin (final concentrations 1: 1000) antibody that, 23 μ l 1x HEPES buffer solutions and 50 μ l are serially diluted.To Immulon 2B flat board (Thermo Electron the human plasma of 50 μ l merging is added in hole).Add the antibody mixing that 50 μ l are prepared to Immulon 2b flat boards Thing, the blood coagulation that 405nm was measured per 15 seconds with dynamics plate reader develops (coagulation development), totally 25 minutes. By the increase plotted over time of optical density, and calculate the clotting time (t1/2).By the mapping of clotting time versus antibody concentration.Make The IC50 of the suppression blood coagulation induced from the result calculating antibody by nonlinear regression analysis with GraphPad Prism.

Fig. 9 shows that antibody 044,087 and 111 does not suppress the blood coagulation that TF is induced, and all other antibody then suppresses.

Table 9 shows that TF- specific humans monoclonal antibody suppresses the IC50 values of blood coagulation.

Table 9-TF- specific humans monoclonal antibody suppresses the IC50 values of blood coagulation.

Embodiment 20

The cell-mediated cytotoxicity of antibody-dependant

The preparation of target cell

Collect the target cell (5x10 of expression TF⁶Individual Bx-PC3 cells, MDA-MB-231 cells or A431 cells), cleaning (cleaning 2 times in PBS, 1500rpm, 5min), and collect in the culture mediums of 1ml RPMI 1640, supplement in the culture medium Reinforced calf serum (cosmic calf serum), Sodium Pyruvate, Pidolidone, MEM NEAA and penicillin/streptomycin, And add 100 μ Ci⁵¹Cr (chromium -51；Amersham Biosciences Europe GmbH,Roosendaal,The Netherlands).37 DEG C in mixture earthquake water-bath are incubated 1 hour.Cleaning cell (clean 2 times in PBS, 1500rpm, After 5min), by Cell resuspension in the medium, and by trypan-blue exclusion living cell counting.Viable cell concentrations are adjusted to 1x10⁵Cell/ml.

The preparation of effector cell：

With standard Ficoll density centrifugation according to operation instruction (the separation of lymphocytes medium of manufacturer；Lonza, Verviers, France) from fresh buffycoat (buffy coat) (Sanquin, Amsterdam, The Netherlands) point From PMNC (PBMCs).By Cell resuspension in the medium after, by trypan-blue exclusion count cell, and Concentration is adjusted to into 1x10⁷Cell/ml.

ADCC is arranged：

By 50 μ l⁵¹The target cell of Cr- marks is transferred in microtiter wells, and is added 50 μ l and be serially diluted and (be diluted in culture In base) antibody.Incubate cells (RT, 15min), and add 50 μ l effector cells, the effector for obtaining and the ratio of target For 100:1.In order to determine maximum cracking level, add 100 μ l 5%Triton-X100 substitution effects thing cells；In order to determine Spontaneous lysis level, adds 100 μ l culture mediums；In order to determine the cracking level for being independent of antibody, add 50 μ l effector cells With 50 μ l culture mediums.Subsequently, at 37 DEG C, 5%CO₂Incubate cells are overnight.After cell centrifugation is settled (1200rpm, 3min), will 75 μ l supernatants are transferred in MICRONIC pipes.Count what is discharged in γ readers⁵¹Cr, such as calculating antibody mediation according to the following formula Cracking percentage：

((cpm sample-cpm are independent of the cracking of antibody)/(the maximum cracking-cpm Spontaneous lysis of cpm)) x100%

Wherein cpm is count per minute (counts per minute).

Figure 10 shows, all cracking of the TF- people's monoclonal antibodies of test induction of ADCC to Bx-PC3 cells, although effect is not With (EC50).

Table 10 shows EC50 value (nM) of the TF- specific humans monoclonal antibody to different clones ADCC.

EC50 value (nM) of the table 10-TF- specific humans monoclonal antibody to different clones ADCC.

Embodiment 21

Complement deposit

Depositions of the complement fragment C3c and C4c on the target cell incubated with TF- people's monoclonal antibody is measured by facs analysis. The target cell (Bx-PC3 or MDA-MB-231 cells) of expression TF is seeded in into 96 hole round bottoms in the RPMI containing 1%BSA to put down In plate (1x10e5 cells/well).Addition antibody (30 μ g/mL), Incubate cells 15 minutes under RT.Add 25 μ L and merge human blood Clearly as complement source, determine that spontaneous complement is combined using heat-inactivated human serum.Cell is incubated 45 minutes at 37 DEG C.Cleaning cell 1 time, and incubate with anti-human C3c FITC or anti-human C4c FITC (DAKO) in FACS buffer solution, and in 30 points of incubated on ice Clock.Sample is analyzed with FACS Canto.

Figure 11 shows, C3c or C4c can not be induced in BxPC3 or MDA-MB-231 cells from the antibody of cross-blocks group I On deposition.All antibody from cross-blocks group II of test induce C3c and C4c depositions, from cross-blocks group III Antibody it is same, but except antibody 003.

Embodiment 22：

Affinity/affinity research

Affinity determines

The knot of antibody and TF is analyzed by surface plasma body resonant vibration in BIAcore 3000 (GE Healthcare) Close.It is analyzed using TFECDHis.It is according to the experimental program that manufacturer is recommended that people's antibody mab (500 resonance units) is solid It is scheduled on CM-5 sensor chips.In brief, after with EDC and NHS surface actives, people's antibody mab is expelled to into activation On CM-5 surfaces, wherein antibody is placed in the sodium acetate of 10mM pH 4.0-5.5, and speed is 5 μ l/min, subsequently uses 1M monoethanolamines Deactivate.The TFECDHis of the series concentration being dissolved in HBS-EP buffer solutions is expelled on fixed antibody, flow velocity is 30 μ l/ Min, continues 180 seconds.Enter pedestrian's monoclonal antibody surface again by injecting 10mM glycine-HCl pH 2.0 or 10mM sodium acetates pH 3.0 It is raw.With double reference minusings (double reference subtraction) and model 1:1 (Lang Gemiaoer) binding analysis enter Action mechanical analysis.

Table 11 shows, for most people monoclonal antibody, measured affinity is in (Asia) nanomolar range.But it is not from complete Portion's antibody can determine kinetic parameter.044 there is high dissociation rate (kd) to be deteriorated really, and with high residual error (residual), it means that curve matching is bad.098th, 111 and 087-Lg6 dissociation rate is too high, so that Biacore 3000 can not measure.

N.a. can not assess=>10^-3sec^-1

Reactive kinetic constant-affinity measurement of the table 11.TF- people monoclonal antibody to TFECDHis

Affinity is determined

The measure that TF (TFECDHis) is combined with the special people's monoclonal antibodies of TF- basically described above, wherein TFECDHis is consolidated (300 resonance units) are scheduled on CM-5 sensor chips, using people's antibody mab of series concentration dynamic analysis is carried out.It is dynamic Mechanical analysis uses double references subtractive (double reference subtraction) and model 1:1 (Lang Gemiaoer) is combined and divided Analyse to carry out.

Table 12 shows the avidity measurements of antibody 11,98,109 and 111.Although 98 and 111 affinity measurement As a result the high dissociation rate (determination limit (i.e. more than Biacore is shown>10^-3)), affinity is determined and then shows nanomole In the range of interaction.

Packet	People monoclonal antibody TF	Affinity nM
			II	11	0.47
II/III	98	4.85
			III	109	0.01
III	111	0.11

Kinetic constants of the table 12.TFECDHIS to TF- people's monoclonal antibody-affinity measurement reaction

Embodiment 23：

The immunohistochemical analysis combined with health adult tissue and pancreatic neoplasm

The combination of people's tissue (colon, heart, kidney, skin, lung and brain) of TF- people's monoclonal antibody and various known expression TF is led to Cross immunohistochemistry (IHC) to be measured.

IHC to freezing tissue

Cutting frozen tissue section (4-6 μ m-thicks), and fix in acetone.Closing endogenous tissue peroxidase (PO), And with normal human serum precincubation histotomy, non-specific knot of the antibody applied below with removing to endogenous Fc acceptors Close.To be applied organizationally with optimum dilution degree for the mouse antibodies (and Negative control mice antibody) of people TF, subsequently used Powervision-PO (goat anti mouse/- rabbit igg)-PO is detected.People's monoclonal antibody of TF specificity is anti-with Fab' goats Human IgG (Fc)-FITC is coupled, and is applied to the histotomy of freezing with 3 dilution factors afterwards, including one predetermined optimal Dilution factor.Subsequently, people's monoclonal antibody-Fab-FITC complexs are detected by rabbit anti-FITC and Powervision-PO.Use AEC conducts Substrate makes PO activity visualizations, nucleus be visualized with haematine.Dyeing is analyzed by bright-field microscope.

The IHC of mouse antibodies is used in the tissue of formalin fix and FFPE (FFPE)

FFPE organizes biopsy with 4 μm of sections, dewaxing, closes endogenous tissue peroxidase, and carries out antigen recovery (retrieval) (pH6, citrate buffer solution).With mouse antibodies incubate before, by histotomy in normal human serum pre-temperature Educate, to prevent the non-specific binding with endogenous Fc acceptors.By for people TF mouse antibodies (and Negative control mice antibody) with Optimum dilution degree is applied to histotomy, is subsequently detected with Powervision-PO (goat anti mouse/- rabbit igg)-PO. PO activity is visualized with AEC as substrate, and nucleus is visualized with haematine.Dyeing is analyzed by bright-field microscope.

Figure 12 shows antibody 013 (positive staining), 011 (positive staining), 114 (positive stainings) and 111 (centre dyes Color) and glomerulus combination example.Antibody 098 and 044 is not bound with glomerulus.

Table 13 gives the general view of coloration result of whole TF- people's monoclonal antibodies in all detected tissues of people's kidney.

The IHC dyeing of the people's glomerulus of table 13.

Table 14 gives selected TF- specific humans monoclonal antibody in people's kidney, colon, heart, brain and skin and human pancreas Coloration result in tumour

Ab

People's kidney

People's colon

Human heart

People's brain

Application on human skin

Pancreatic neoplasm

13

Malpighian corpuscle+

Basilar memebrane ++

-

+

Epidermis+

+++

114

Malpighian corpuscle ++

Basilar memebrane ++

-

++

Epidermis ++

++++

11

Malpighian corpuscle+

Basilar memebrane ++

-

++

n.a.(+)

+++

44

-

Basilar memebrane+

-

+/-

n.a.

++

98

-

Basilar memebrane+

-

+/-

n.a.(+)

+++

111

Malpighian corpuscle is +/-

Basilar memebrane+

-

+

n.a.

+++

The IHC dyeing of the health adult tissue of table 14. and pancreatic neoplasm

TF- people's monoclonal antibody shows that all TF- people's monoclonal antibodies have positive staining (Figure 13 with the IHC of human pancreas' tumor combination analyses Illustrate).

Embodiment 24：

The treatment of the MDA-MB-231 tumor xenogeneic grafts to having set up in SCID mice mammary fat pad

The body of TF- people's monoclonal antibody is determined in the MDA-MB-231 xenograft tumors in situ set up in SCID mice body Interior effect.By 2x10⁶The individual cancer cell subcutaneous being placed in PBS are injected in the mammary fat pad of Female SCID mice second, with Start afterwards to be treated with TF- people's monoclonal antibody or control monoclonal antibody (people monoclonal antibody-KLH) when tumor size becomes and can measure.Antibody In the 21st day (260 μ g/ mouse), 28 days (130 μ g/ mouse) and 42 days (130 μ g/ mouse) injection.Gross tumor volume is at least weekly Measurement 2 times.Volume (mm is calculated by slide calliper rule (PLEXX) measurement result³)：0.52x (length) x (width)²。

Figure 14 shows that antibody 114,111,013,098,011 and 044 all can effectively suppress the original position having built up MDA-MB-231 tumour growths.

Embodiment 25：

Tentative repeat administration (Pilot repea tdosing) of the TF- specific human monoclonal antibodies in machin

In order to obtain preliminary information toxicologic with regard to TF- specific human monoclonal antibodies, including assessment antibody interference coagulation cascade And therefore potentially increase exposure animal bleeding risk ability, the administration of tentative repeated doses has been carried out in machin and has been ground Study carefully.

Two males and two female cynomolgus monkeys (Macaca fascicularis), the age is of about 2 years, receives vein Interior injection of antibodies 011：

- research the 1st day：0mg/kg (only solvent (vehicle))

- the 8 day：1mg/kg；1mL/ minutes

- the 15 day：10mg/kg；1mL/ minutes

- the 22 day：100mg/kg；1mL/ minutes

Tracking animal made animal euthanasia up to 27 days in the time point, carried out autopsy and organ histology's assessment.

Main research terminal is：

- clinical observation:Determine from gum, the sign of ophthalmorrhagia daily.

- functional bleeding the time and lose blood:The 1st, 8,15 and 22 days (1,24 and 120h after administration) and before 2 tests Time point is measured.

- blood/blood vestige/clot：All tissue carries out HE dyeing (tissue obtained in final putting to death determines)

Blood in-urine, excrement, vomitus：Daily/determine weekly.

Repeat or the antibody 011 of increasing dosage administration is not observed apparent toxicity.Animal does not show clinical signs, does not have There is the sign of cytokine release.Additionally, not showing the apparent clinical indication of the impaired or systemic bleeding of blood coagulation system.Giving The time point of 1 hour after medicine, the average bleeding time of the 22nd day is considerably longer than the result (p=0.012) observed at the 1st day. 8th, 15 and 22 days compared with the 1st day, without other differences statistically significantly.Further, it was found that there is no table to major organs See toxicity and affect without unfavorable hematology.The preliminary conclusion of the Histological assessment with regard to organizing obtained from the research It is, without the histology discovery that can be processed as tester with attribution in 4 processed animals.

Figure 15 shows each data point (two repeat samples) of every animal, used as the function of time.The 1st, 8, The bleeding time of 4 animals of (pre-trial) time point determining before 15 and 22 days (1,24 and 120h) and two tests.

Embodiment 26

Prevention and treatment property processes BxPC3 tumor xenogeneic grafts in SCID mice

Determine TF- people's monoclonal antibody prevention and treatment property process SCID mice in BxPC3 cell xenografts it is internal Effect.By 10x10⁶The individual BxPC3 cancer cell subcutaneous being placed in PBS are injected in Female SCID mice body, subsequently use TF- people Monoclonal antibody or control monoclonal antibody (people monoclonal antibody-KLH) are treated.It is anti-in tumor inducing pneumoretroperitoneum injection in 1 hour for prophylactic treatment Body (400 μ g/ mouse).For therapeutic treatment, injection of antibodies (300 μ g/ mouse) is started after 8 days in tumor inducing, it is subsequently every Zhou Jinhang antibody injects (150 μ g/ mouse).Gross tumor volume is at least determined 2 times weekly.Volume (mm³) measured by slide calliper rule (PLEXX) As a result calculate：0.52x (length) x (width)²。

Figure 16 shows that TF special property people monoclonal antibody being capable of preventative and therapeutic treatment BxPC3 xenograft tumours.

Embodiment 27

DNA reorganization between mouse and people TF, to determine to reference to the important domain of anti-TF people's monoclonal antibody

In order to determine the domain important to the combination of anti-TF people's monoclonal antibody and people TF, carry out DNA between people and mouse TF and change Group.The domain of people is replaced by domain with mouse and prepares reorganization construct from the DNA of encoding human TF, and by with people domain replace mouse domain from The DNA of coding mouse TF prepares reorganization construct.If combination of certain domain in people TF to anti-TF people's monoclonal antibody is important, then when When being replaced with mouse domain, combination will be lost.People and mouse TF have 57% homology in protein level.Figure 17 A and 17B show and contain There are people's TF constructs (TFhs, containing TFmm domains) in mouse TF domains, and the mouse TF constructs in the domains of TF containing someone.With construct or list Only carrier (pcDNA3.3SP；Simulation) transient transfection HEK293F cells.Facs analysis are carried out basically described above, are used The parent material of 30 μ g/mL purifying.People's monoclonal antibody-KLH is used as control antibodies.

Figure 17 shows, in addition to one, whole anti-TF people's monoclonal antibodies are all only combined with people TF, and are not combined with mouse TF.People Monoclonal antibody-TF-003 shows some combinations with mouse TF.

Figure 18 A-O show the combination result of different anti-TF people's monoclonal antibodies and the construct expressed on HEK293F cells. These results are summarized in table 15.In the table, according to domain important to the combination of these people's monoclonal antibodies on people TF by these anti-TF People's monoclonal antibody is grouped.

Table 15

Embodiment 28

The combination (being determined by ELISA) of the Fab fragments of anti-TF people's monoclonal antibody and TF extracellular domains and with BxPC3 cells on The combination (being determined by FACS) of cell TF

The Fab of anti-TF people's monoclonal antibody is measured by ELISA (extracellular domain of coating TF) and FACS (TF on BxPC3 cells) The combination of fragment and TF.ELISA is substantially carried out as mentioned before.With reference to donkey-anti-human H+L for being coupled with HRP of Fab fragments enter Row detection.Facs analysis are substantially carried out as mentioned before.Using FITC be coupled goat anti-human IgG (H+L) (Jackson) The leading candidate that detection is combined.Fluorescence is measured on FACSCantoII.The analysis of binding curve GraphPad Prism 5 Software is carried out as previously mentioned.

Figure 19 shows, compared with people's monoclonal antibody-TF-011Fab fragments, people's monoclonal antibody-TF-098 and -111Fab fragments show with The combination less (being measured by ELISA) of TF extracellular domains.

Figure 20 shows, compared with people's monoclonal antibody-TF-011Fab fragments, people's monoclonal antibody-TF-098 and -111Fab fragments show with The combination less (being measured by the FACS to BxPC3 cells) of cell TF.

Table 16 show the combination EC50 values (by ELISA determinations) of people's monoclonal antibody-TF Fab fragments and TF extracellular domains and with it is thin The combination EC50 values (being measured by the FACS of BxPC3 cells) of born of the same parents TF.

The combination EC50 values (being determined by ELISA) of table 16- people's monoclonal antibody-TF Fab fragments and TF extracellular domains and and BxPC3 The general view of the EC50 values (being determined by FACS) of the combination of cell TF on cell.

EC50 values are in terms of μ g/mL.

Na- can not be calculated.

Embodiment 29

The combination of the clone of anti-TF monoclonal antibodies and expression varying level TF

The knot of film combination TF in clone of the anti-TF people's monoclonal antibody with expression varying level TF is determined by facs analysis Close, substantially as mentioned before.Anti-mouse IgGFc that positive control is coupled using the follow-up PE- of the anti-TF antibody of mouse.Fluorescence exists Measure on FACSCantoII.The analysis of binding curve is substantially carried out as previously mentioned with the softwares of GraphPad Prism 5. The amount of TF molecules passes through operation instructions of the Qifi kits (Dako, Glostrup, Denmark) according to manufacturer in clone Determined.Measure SW480 cells each cells and express～20,000 TF molecule, SK-OV-3 cells each cell expression～ 60,000 molecules, AsPC-1 cells each cell expresses～175,000 molecule, and MDA-MB-231 cells each cell tables Up to～900,000 molecules.

In clone MDA-MD-231 of high expression TF, people monoclonal antibody-TF-98 and -111 shows and people monoclonal antibody-TF- Figure 21 11, -13 and 109 similar binding characteristics.In the relatively low clone of each cell TF molecular numbers, such as SK-OV-3 and SW480 are thin In born of the same parents system, people monoclonal antibody-TF-98 and 111 shows the binding characteristics different from other people's monoclonal antibody-TF antibody.

Claims

1. a kind of human antibody of combination human tissue factor.

2. the antibody of claim 1, wherein when being measured with the assay method described in embodiment 13, the antibody is with 3nM Or lower, such as 0.50nM or lower, such as 0.35nM or lower, such as 0.20nM or lower, such as 0.1nM or lower table See affinity (EC₅₀) combined with the extracellular domain of tissue factor.

3. the antibody of any one of aforementioned claim, the wherein antibody combine the mammalian cell of the expression tissue factor, example The A431 cells for such as being transfected with the construct of encoding tissue factor, it is preferable that when being entered with the assay method described in embodiment 14 When row is determined, its apparent affinity (EC₅₀) be 10nM or lower, such as 8nM or lower, such as 5nM or lower, such as 2nM or It is lower, such as 1nM or lower, such as 0.5nM or lower, such as 0.3nM or lower.

4. the antibody of any one of aforementioned claim, wherein antibody can induce the cell of antibody-dependant in A431 cells Cytotoxicity, it is preferable that when being measured with the assay method described in embodiment 20, its EC₅₀It is worth for 2nM or lower, example Such as 1nM or lower, such as 0.7nM or lower, or 0.3nM or lower, such as 0.20nM or lower, or 0.1nM or lower, Or 0.05nM or lower.

5. the antibody of any one of aforementioned claim, wherein when being measured with the method described in embodiment 24, this resists Body can effectively suppress the growth of the MDA-MB-231 tumours set up, and/or ought be surveyed with the method described in embodiment 26 Regularly, the growth of BxPC3 tumours set up can effectively be suppressed.

6. the antibody of any one of aforementioned claim, the wherein blood coagulation of antibody suppression tissue factor induction, it is preferable that when with Such as when assay method described in embodiment 19 is measured, its median inhibitory concentration is less than 10nM, e.g., less than 5nM, little In 2nM, e.g., less than 1nM.

7. the antibody of any one of aforementioned claim, wherein antibody suppression FVIIa is combined with tissue factor, it is preferable that when When being measured with the assay method described in embodiment 15, the maximum suppression value that it suppresses is more than 80%, is greater than 90%.

8. the antibody of any one of aforementioned claim, the IL- of the MDA-MB-231 cells of wherein antibody suppression FVIIa inductions 8 releases, it is preferable that when being measured with the assay method described in embodiment 17, the maximum suppression value of suppression is more than 40%, 50% is greater than, 60% is greater than.

9. the antibody in any one of aforementioned claim, wherein antibody suppression FX is transformed into FXa by TF/FVIIa complexs, Preferably, when being measured with the assay method described in embodiment 18, suppress to be less than 50%, e.g., less than 40%, for example In the range of 1-30%.

10. the antibody of any one of aforementioned claim, wherein the antibody with include comprising sequence SEQ ID NO:9 VH areas With comprising sequence SEQ ID NO:The antibody competition tissue factor in 65 VL areas is combined.